Methods for identifying compounds that alter kinase activity

ABSTRACT

The invention is directed to purified and isolated novel murine and human kinase polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and the uses of the above.

This application claims the priority of provisional application U.S.Ser. No. 60/136,781, filed May 28, 1999, which is incorporated byreference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to purified and isolated novel murine andhuman kinase polypeptides and fragments thereof, the nucleic acidsencoding such polypeptides, processes for production of recombinantforms of such polypeptides, fragmented peptides derived from thesepolypeptides, antibodies generated against these polypeptides, methodsof identifying activators and inhibitors of the activity of thesekinases, and therapeutic and diagnostic uses thereof.

2. Background

Cells respond to external signals and internal signals, such as thoseproduced by disease conditions, by activating cellular signalingpathways. Cellular signaling often involves a molecular activationcascade, during which a receptor propagates a ligand-receptor mediatedsignal by specifically activating intracellular protein kinases whichphosphorylate target substrates. These substrates can themselves bekinases which become activated following phosphorylation.

The eukaryotic protein kinases make up a large and rapidly expandingfamily of proteins related on the basis of homologous catalytic domains.Spurred by the development of gene cloning and sequencing methodologies,distinct protein kinase genes have been identified from a wide selectionof invertebrates and lower eukaryotes, including Drosophila,Caenorhabditis elegans, Aplysia, Hydra, Dictyostelium, and budding(Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe)yeast. Homologous genes have also been identified in higher plants.Protein kinases, however, are not limited to the eukaryotes. Enzymeactivities have been well documented in prokaryotes, but the prokaryoticprotein kinase genes are not obviously similar to those of theeukaryotes.

Given the important function of kinases in general, there is a need inthe art for additional members of the kinase family. In addition, inview of the continuing interest in protein research, the discovery,identification, and roles of new proteins, such as protein kinases, areat the forefront of modem molecular biology and biochemistry. Despitethe growing body of knowledge, there is still a need in the art for theidentity and function of proteins having kinase activities. In addition,because there is an unmet need for therapeutic compounds which modulatekinase activity and because protein kinases are useful biochemicalreagents, there is also need in the art for the continued discovery ofunique members of the protein kinase family and potential therapeutictargets thereof.

SUMMARY OF THE INVENTION

The invention aids in fulfilling these various needs in the art byproviding isolated murine and human kinase nucleic acids andpolypeptides encoded by these nucleic acids. Particular embodiments ofthe invention are directed to isolated murine and human kinase nucleicacid molecules comprising the DNA sequences of SEQ ID NOs:1-7 andisolated murine and human kinase nucleic acid molecules encoding theamino acid sequences of SEQ ID NOs:8-14, as well as nucleic acidmolecules complementary to these sequences. Both single-stranded anddouble-stranded RNA and DNA nucleic acid molecules are encompassed bythe invention, as well as nucleic acid molecules that hybridize to adenatured, double-stranded DNA comprising all or a portion of SEQ IDNOs:1-7. Also encompassed are isolated nucleic acid molecules that arederived by in vitro mutagenesis of nucleic acid molecules comprisingsequences of SEQ ID NOs:1-7, that are degenerate from nucleic acidmolecules comprising sequences of SEQ ID NOs:1-7, and that are allelicvariants of DNA of the invention. The invention also encompassesrecombinant vectors that direct the expression of these nucleic acidmolecules and host cells stably or transiently transformed ortransfected with these vectors.

In addition, the invention encompasses methods of using the nucleicacids noted above to identify nucleic acids encoding proteins havingkinase activity and to study cell signal transduction.

The invention also encompasses the use of sense or antisenseoligonucleotides from the nucleic acid of SEQ ID NOs:1-7 to inhibit theexpression of the polynucleotide encoded by the kinase genes.

The invention also encompasses isolated polypeptides and fragmentsthereof encoded by these nucleic acid molecules including solublepolypeptide portions of SEQ ID NOs:8-14. The invention furtherencompasses methods for the production of these polypeptides, includingculturing a host cell under conditions promoting expression andrecovering the polypeptide from the culture medium. Especially, theexpression of these polypeptides in bacteria, yeast, plant, insect, andanimal cells is encompassed by the invention.

In general, the polypeptides of the invention can be used to studycellular processes such as signal transduction and to screen forcompounds which modulate kinase activity which may have therapeuticpotential. In addition, these polypeptides can be used to identifyproteins associated with the polypeptides of the invention.

In addition, the invention includes assays utilizing these polypeptidesto screen for potential inhibitors of activity associated withpolypeptide counter-structure molecules, and methods of using theseinhibitors as therapeutic agents for the treatment of cancer and otherproliferative diseases and diseases mediated by polypeptidecounter-structure molecules. Further, methods of using thesepolypeptides in the design of inhibitors thereof are also an aspect ofthe invention.

The invention further provides a method for using these polypeptides asmolecular weight markers that allow the estimation of the molecularweight of a protein or a fragmented protein, as well as a method for thevisualization of the molecular weight markers of the invention thereofusing electrophoresis. The invention further encompasses methods forusing the polypeptides of the invention as markers for determining theisoelectric point of an unknown protein, as well as controls forestablishing the extent of fragmentation of a protein.

Further encompassed by this invention are kits to aid in thesedeterminations.

Further encompassed by this invention is the use of the kinase nucleicacid sequences, predicted amino acid sequences of the polypeptide orfragments thereof, or a combination of the predicted amino acidsequences of the polypeptide and fragments thereof for use in searchingan electronic database to aid in the identification of sample nucleicacids and/or proteins.

Isolated polyclonal or monoclonal antibodies that bind to thesepolypeptides are also encompassed by the invention, in addition the useof these antibodies to aid in purifying the murine and human kinasepolypeptides.

Also encompassed by the invention is a method of designing an inhibitorof the kinase polypeptide of the invention, the method comprising thesteps of determining the three-dimensional structure of suchpolypeptide, analyzing the three-dimensional structure for the likelybinding sites of substrates, synthesizing a molecule that incorporates apredicted reactive site, and determining the polypeptide-inhibitingactivity of the molecule.

Another aspect of the invention is a method for identifying compoundsthat activate or inhibit kinase activity comprising:

(a) bringing a test compound into contact with the polypeptide of theinvention and a substrate; and

(b) determining whether the test compound activates or inhibits thekinase activity of said polypeptide. In a preferred embodiment of thismethod, the test compound is brought into contact with the polypeptidein a cell containing at least one recombinant vector that directs theexpression of at least one polynucleotide encoding said polypeptide.

Another aspect of the invention is a method for inhibiting the kinaseactivity of the polypeptide of the invention comprising forming amixture of the polypeptide, a substrate, and a compound, wherein thecompound blocks the binding of the polypeptide with the substrate.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-6 each depict an alignment of the amino acid sequence of akinase of the invention (top line) with the conserved amino acidresidues of the family of protein serine/threonine kinases (bottomline). Invariant residues are shown in UPPER CASE letters,nearly-invariant residues as lower case, conserved hydrophobic residuesas (o), conserved polar residues as (*), and conserved small residueswith near neutral polarity as (+).

FIG. 1 presents the amino acid sequence alignment of the consensusprotein serine/threonine kinase sequence with MDCK-1.

FIG. 2 presents the amino acid sequence alignment of the consensusprotein serine/threonine kinase sequence with MDCK-2.

FIG. 3 presents the amino acid sequence alignment of the consensusprotein serine/threonine kinase sequence with MDCK-3.

FIG. 4 presents the amino acid sequence alignment of the consensusprotein serine/threonine kinase sequence with SK-1.

FIG. 5 presents the amino acid sequence alignment of the consensusprotein serine/threonine kinase sequence with MLSK-2.

FIG. 6 presents the amino acid sequence alignment of the consensusprotein serine/threonine kinase sequence with LNRK-1.

DETAILED DESCRIPTION OF THE INVENTION

The molecules encompassed in the invention include the followingnucleotide and amino acid sequences:

Kinase: DNA Sequence: Protein Sequence: MDCK-1 SEQ ID NO:1 SEQ ID NO:8MDCK-2 SEQ ID NO:2 SEQ ID NO:9 MDCK-3 SEQ ID NO:3 SEQ ID NO:10 MLSK-1SEQ ID NO:4 SEQ ID NO:11 MLSK-2 SEQ ID NO:5 SEQ ID NO:12 ss4694 SEQ IDNO:6 SEQ ID NO:13 LNRK-I SEQ ID NO:7 SEQ ID NO:14 LNRK-1 primers SEQ IDNO:15 and SEQ ID NO:16

The discovery of the nucleic acids of the invention enables theconstruction of expression vectors comprising nucleic acid sequencesencoding polypeptides; host cells transfected or transformed with theexpression vectors; isolated and purified biologically activepolypeptides and fragments thereof; the use of the nucleic acids oroligonucleotides thereof as probes to identify nucleic acid encodingproteins having kinase activity; the use of single-stranded sense orantisense oligonucleotides from the nucleic acids to inhibit expressionof polynucleotides encoded by the kinase genes of the invention; the useof such polypeptides and soluble fragments to function as kinases; theuse of such polypeptides and fragmented peptides as molecular weightmarkers; the use of such polypeptides and fragmented peptides ascontrols for peptide fragmentation, and kits comprising these reagents;the use of such polypeptides and fragments thereof to generateantibodies; and the use of antibodies to purify the human and murinekinase polypeptides.

Nucleic Acid Molecules

In a particular embodiment, the invention relates to certain isolatednucleotide sequences that are free from contaminating endogenousmaterial. A “nucleotide sequence” refers to a polynucleotide molecule inthe form of a separate fragment or as a component of a larger nucleicacid construct. The nucleic acid molecule has been derived from DNA orRNA isolated at least once in substantially pure form and in a quantityor concentration enabling identification, manipulation, and recovery ofits component nucleotide sequences by standard biochemical methods (suchas those outlined in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd sed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989)). Such sequences are preferably provided and/or constructedin the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, that are typically present ineukaryotic genes. Sequences of non-translated DNA can be present 5′ or3′ from an open reading frame, where the same do not interfere withmanipulation or expression of the coding region.

Nucleic acid molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using the cDNA ofSEQ ID NOs:1-7, or a suitable fragment thereof, as a probe.

The DNA molecules of the invention include full length genes as well aspolynucleotides and fragments thereof. Other embodiments include DNAencoding a soluble form of the protein.

The nucleic acids of the invention are preferentially derived frommurine and human sources, but the invention includes those derived fromother sources, as well.

Preferred Sequences

Particularly preferred nucleotide sequences of the invention are SEQ IDNOs:1-7, as set forth above. The sequences set forth in SEQ ID NOs:1-3are Murine Dendritic Cell Kinases (MDCK) 1 to 3, respectively. SEQ IDNOs:4-5 are Murine Lymph node Stromal cell Kinases (MLSK) 1 and 2.Finally, SEQ ID NO:6 is from human dendritic cells and is calledSS-4694. This sequence was utilized to clone full length SEQ ID NO:7,called Large NIK-Related Kinase (LNRK) 1.

Clones having the nucleotide sequences of SEQ ID NOs:1-7 were isolatedas described in Example 1. The sequences of amino acids encoded by theDNAs of SEQ ID NOs:1-7 are shown in SEQ ID NOs:8-14. As set forth indetail below, the amino acid sequences for MDCK-1 (SEQ ID NO:8), MDCK-2(SEQ ID NO:9), MDCK-3 (SEQ ID NO:10), MLSK-1 (SEQ ID NO:11), MLSK-2 (SEQID NO:12), ss4694(SEQ ID NO:13), and LNRK-1 (SEQ ID NO:14) identify thepolynucleotides as a member of the kinase superfamily.

Particularly preferred polynucleotides that encode the kinases of theinvention and which comprise certain nucleotide sequences of SEQ IDNOs:1-7 are as follows:

nucleotides 71-893 of SEQ ID NO:1 (MDCK-1);

nucleotides 115-1020, 136-1020, and 190-1020 of SEQ ID NO:2 (MDCK-2);

nucleotides 243-1307 of SEQ ID NO:3 (MDCK-3);

nucleotides 123-2015 of SEQ ID NO:4 (MLSK-1);

nucleotides 121-1053 of SEQ ID NO:5 (MLSK-2);

nucleotides 95-892 of SEQ ID NO:6 (ss4694); and

nucleotides 1-4080 of SEQ ID NO:7 (LNRK-1).

Additional Sequences

Due to the known degeneracy of the genetic code, wherein more than onecodon can encode the same amino acid, a DNA sequence can vary from thatshown in SEQ ID NOs:1-7 and still encode a polypeptide having the aminoacid sequence of SEQ ID NOs:8-14. Such variant DNA sequences can resultfrom silent mutations (e.g., occurring during PCR amplification), or canbe the product of deliberate mutagenesis of a native sequence.

The invention thus provides isolated DNA sequences encoding polypeptidesof the invention, selected from: (a) DNAs comprising the nucleotidesequence of SEQ ID NOs:1-7; (b) DNAs encoding the polypeptides of SEQ IDNOs:8-14; (c) DNA capable of hybridization to a DNA of (a) or (b) underconditions of moderate stringency and which encodes polypeptides of theinvention; (d) DNA capable of hybridization to a DNA of (a) or (b) underconditions of high stringency and which encodes polypeptides of theinvention, and (e) DNA which is degenerate as a result of the geneticcode to a DNA defined in (a), (b), (c), or (d) and which encodepolypeptides of the invention. Of course, polypeptides encoded by suchDNA sequences are encompassed by the invention.

As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), and includeuse of a prewashing solution for the nitrocellulose filters 5× SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50%formamide, 6× SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5× SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2× SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe. Preferred hybridizing polynucleotides are thosethat are at least 25%, more preferably 50%, and most preferably 75% ofthe length of the polynucleotide to which they hybridize.

Also included as an embodiment of the invention is DNA encodingpolypeptide fragments and polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), as described below.

In another embodiment, the nucleic acid molecules of the invention alsocomprise nucleotide sequences that are at least 80% identical to anative sequence. Also contemplated are embodiments in which a nucleicacid molecule comprises a sequence that is at least 90% identical, atleast 95% identical, at least 98% identical, at least 99% identical, orat least 99.9% identical to a native sequence.

The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The preferreddefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)for nucleotides, and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

The invention also provides isolated nucleic acids useful in theproduction of polypeptides. Such polypeptides may be prepared by any ofa number of conventional techniques. A DNA sequence encoding a kinasepolypeptide of the invention, or desired fragment thereof, may besubcloned into an expression vector for production of the polypeptide orfragment. The DNA sequence advantageously is fused to a sequenceencoding a suitable leader or signal peptide. Alternatively, the desiredfragment may be chemically synthesized using known techniques. DNAfragments also may be produced by restriction endonuclease digestion ofa full length cloned DNA sequence, and isolated by electrophoresis onagarose gels. If necessary, oligonucleotides that reconstruct the 5′ or3′ terminus to a desired point may be ligated to a DNA fragmentgenerated by restriction enzyme digestion. Such oligonucleotides mayadditionally contain a restriction endonuclease cleavage site upstreamof the desired coding sequence, and position an initiation codon (ATG)at the N-terminus of the coding sequence.

The well-known polymerase chain reaction (PCR) procedure also may beemployed to isolate and amplify a DNA sequence encoding a desiredprotein fragment. Oligonucleotides that define the desired termini ofthe DNA fragment are employed as 5′ and 3′ primers. The oligonucleotidesmay additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto an expression vector. PCR techniques are described in Saiki et al.,Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds.,Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols:A Guide to Methods and Applications, Innis et al., eds., Academic Press,Inc. (1990).

Polypeptides and Fragments Thereof

The invention encompasses polypeptides and fragments thereof in variousforms, including those that are naturally occurring or produced throughvarious techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

Polypeptides and Fragments Thereof

The polypeptides of the invention include full length proteins encodedby the nucleic acid sequences set forth above. Particularly preferredpolypeptides comprise the amino acid sequences of SEQ ID NOs:8-14 asfollows:

amino acids 1-275, 25-275, 140-226, and 149-175 of SEQ ID NO:8 (MDCK-1);

amino acids 1-302, 8-302, 26-302, 34-295, 60-229, 157-183, and 198-229of SEQ ID NO:9 (MDCK-2);

amino acids 1-355, 23-279, 124-221, 134-169, and 183-214 of SEQ ID NO:10(MDCK-3);

amino acids 1-631, 57-309, 129-254, 175-200, and 215-247 of SEQ ID NO:11(MLSK-1);

amino acids 1-311, 14-271, 41-213, 124-161, and 175-206 of SEQ ID NO:12(MLSK-2);

amino acids 1-266, 1-261, 25-258, 101-233, 149-175, and 190-226 of SEQID NO:13 (ss4694); and

amino acids 1-1360, 25-306, 25-258, 101-233, 149-175, and 190-226 of SEQID NO:14 (LNRK-1).

As set forth in FIGS. 1-6, alignments of the polypeptide sequences ofthe invention (SEQ ID NOs:8-12 and 14) with the conserved residues ofthe family of protein serine/threonine kinases indicate that thepolypeptides of the invention are serine/threonine kinases. The homologystudies described below for each of these six sequences provideadditional support for their activity as serine/threonine kinases.

MDCK-1

As noted above, three of the cDNAs (MDCK-1, MDCK-2 and MDCK-3) wereisolated from murine (Mus musculus C57 Black6) dendritic cells. MDCK-1(SEQ ID NO:1) encodes an open reading frame (nucleotides 71-1009) whichcommences with an ATG coding for a methionine residue and ends with anin-frame stop codon. In addition, the predicted polypeptide of MDCK-1(SEQ ID NO:8, 275 amino acids) contains a canonical proteinserine/threonine kinase domain from amino acid residues 25 to 296.

In initial searches of public databases using the BLAST algorithm, itwas found that MDCK-1 closely resembles the following GenBank entries:Accession #AF096300, HPK/GCK-like kinase (HGK) [Homo sapiens] (85% aminoacid identity, 248 out of 291 amino acid residues); Accession #U88984,Nck-interacting kinase (NIK) [Mus musculus] (83% amino acid identity,243 out of 291 amino acid residues); Accession #AB01123, KIAA0551protein [Homo sapiens] (85% amino acid identity, 225 out of 264 aminoacid residues). A search of the Derwent GeneSeq patent database usingthe BLAST algorithm revealed: Human protein kinase SOK-1 (Ste20 oxidantstress response kinase protein) which has 46% amino acid identity withMDCK-1 and Human protein kinase HPK, which has 44% amino acid identitywith MDCK-1. In the kinase domain, the strongest homology of MDCK-1 wasfound to be with human HPK/GCK-like kinase HGK (Accession AF096300, 85%amino acid identity) and murine NIK (Accession U88984, 83% amino acididentity) although there are close similarities with a number of othermembers of a growing subfamily of kinases collectively termed theGC-kinases (for Germinal Center Kinases).

In more recent searches of public databases using BLAST, the followingGenbank protein sequences were found to have even greater sequencesimilarity to the MDCK-1 polypeptide: Accession #AB035697,Misshapen/NIKs-related kinase MINK-1 [Mus musculus] (100% amino acididentity, 275 of 275 residues); Accession #AB041925, GCK family kinaseMINK-2 [Mus musculus] (100% amino acid identity, 275 of 275 residues);Accession #AB035698, Misshapen/NIKs-related kinase MINK-1 [Homo sapiens](99% amino acid identity, 274 of 275 residues); Accession #AB041926, GCKfamily kinase MINK-2 [Homo sapiens] (99% amino acid identity, 274 of 275residues). MINK is a novel GCK family kinase that is most abundant inbrain. Its expression is up-regulated in the course of the postnataldevelopment of mouse cerebrum. MINK is an upstream activator of thestress-activated protein kinase cascade. MINK-2 is an alternativelyspliced form of MINK-1; a splicing cassette consisting of 24 bp encodinga 6 amino acid polypeptide is inserted. MINK-2 is more abundant thanMINK-1 in mouse brain. The search of the Derwent GeneSeq patent databaseusing the BLAST algorithm revealed: Accession #Y55931, Human ZC1 protein(91% amino acid identity, 251 of 275 residues); Accession #Y55954, MouseSTE20-related protein kinase NIK protein (89% amino acid identity, 246of 275 residues); Accession #Y55933, Human ZC3 protein (98% amino acididentity, 236 of 240 residues); Accession #Y55953, NematodeSTE20-related protein kinase ZC504.4 protein (82% amino acid identity,221 of 268 residues; and Accession #Y55932, Human ZC2 protein. protein(90% amino acid identity, 216 of 239 residues).

MDCK-1 is clearly a member of the GCK family which includes the NcKinteracting kinase family (NIK). GCK kinases are a subfamily of theSterile20 (STE-20) protein kinases. This family of kinases are involvedas upstream activators of the Jun terminal kinases (JNK's) which arecharacteristically activated in response to a variety of cellularstresses. The properties of these kinases were recently reviewed[Kyriakis J M, 1999, Signaling by the germinal center kinase family ofprotein kinases, J. Biol. Chem. 274(9): 5259-62; which is incorporatedby reference herein]. The sequence of the MDCK-1 polypeptide indicatesthat it may be a truncated version of murine MINK; however, MDCK-1protein contains an intact kinase domain and is predicted to form acatalytically functional kinase when expressed in host cells, andtherefore can be used to modulate or regulate these pathways in diseaseconditions, and to identify activators and inhibitors of these pathways.

MDCK-2

The MDCK-2 cDNA was isolated from murine dendritic cells. Nucleotides115-1020 of MDCK-2 (SEQ ID NO:2) encode an open reading frame with threepossible in-frame initiator methionines at amino acid residues 1, 8, and26. All three of these methionine residues are upstream of the firstkinase subdomain. Any one or more of them could be the physiologicallyrelevant initiator Met; however, amino acid sequence similarity to humanNEK5 protein suggests that the methionine at the first position is theinitiator Met residue. SEQ ID NO:9 shows the open reading frame with apredicted polypeptide of 304 amino acids. Like MDCK-1, MDCK-2 contains acanonical protein serine/threonine kinase domain (amino acid residues34-295 of SEQ ID NO:9).

In initial searches of the public databases using the BLAST algorithm itwas found that MDCK-2 most closely resembles a GenBank entry: Accession#Z50873. MDCK-2 demonstrated 77% amino acid amino acid identity (189 outof 245 residues) to this putative kinase from Caenorhabditis elegans.MDCK-2 also appeared related to the NEK (NIMA-related protein kinase)family of kinases. A BLAST search of the Derwent patent databaseindicated a human kinase called HPK-1 has 85% amino acid amino acididentity with MDCK-2 protein over a 232 amino acid span.

In more recent searches of the public databases using BLAST, thefollowing Genbank protein sequence was found to have sequence similarityto the MDCK-2 polypeptide: Accession #AB026289, protein kinase SID6-1512[Homo sapiens] (81% amino acid identity, 238 of 291 residues). Thesearch of the Derwent GeneSeq patent database using the BLAST algorithmrevealed: Accession #Y59143, Human serine/threonine kinase, NEK5 protein(97% amino acid identity, 295 of 302 residues). MDCK-2 appears to be themurine homologue of human NEK5, and there are several other NEKs thatare closely related to MDCK-2. The NEKs were named based on theirrelationship with the NIMA family (NIMA-related kinase), which areproteins that regulate mitosis and mitogenesis.

MDCK-3

MDCK-3 was isolated from murine dendritic cells and encodes an openreading frame of 355 amino acids (nucleotides 243-1310 of SEQ ID NO:3)which contains a canonical serine/threonine kinase domain (amino acidresidues 23-279 of SEQ ID NO:10 and FIG. 3).

Initial searches of available protein sequence databases revealed thatMDCK-3 had not previously been described. The closest homolog in thesedatabases was rat calcium/calmodulin-dependent kinase 1 (Genbankaccession Q63450) which shares 77.7% sequence amino acid identity withMDCK-3.

In more recent searches of the public databases using BLAST, thefollowing Genbank protein sequences were found to have sequencesimilarity to the MDCK-3 polypeptide: Accession #AAA66944, CaM-likeprotein kinase [Rattus norvegicus] (84% amino acid identity, 266 of 314residues); Accession #AAA99458, cam kinase I [Homo sapiens] (84% aminoacid identity, 265 of 314 residues); and Accession #AAA19670, proteinkinase I [Rattus norvegicus] (83% amino acid identity, 263 of 314residues). The search of the Derwent GeneSeq patent database using theBLAST algorithm revealed additional calcium/calmodulin-dependent proteinkinases as having similarity to MDCK-3, but none that shared more than70% amino acid sequence amino acid identity with MDCK-3. From theseresults it is clear that MDCK-3 is a member of theCa2+/calmodulin-dependent kinase family.

MDCK-3 RNA has been found to be expressed in certain cancer cell lines,but not in others (see the table below); thus, MDCK-3 may be useful as amarker for the presence certain cancer types, such as colon cancer orovarian cancer.

Cancer Cell Line: Cancer Type: MDCK-3 Expression: Colo205 coloncarcinoma + HT29 colon carcinoma + IGROV-1 ovarian carcinoma + MDA231breast adenocarcinoma + Jurkat T-cell leukemia − MALME-3M melanoma − WM9melanoma − WM-35 melanoma − WM164 melanoma − WM-3211 melanoma +

MDCK-3 RNA has also been demonstrated to be regulated in response todendritic cell maturation and/or activation, as described in Example 7.

MLSK-1

Two additional clones, MLSK-1 and MLSK-2, were isolated from murinelymph node stromal cells. MLSK-1 (SEQ ID NO:11) contains an open readingframe encoded by nucleotides 123-2015 of SEQ ID NO:4, which commenceswith an ATG coding for a methionine residue and ends with an in-framestop codon. The predicted 631 amino acid sequence (SEQ ID NO:11)contains a canonical protein serine/threonine kinase domain at residues57 to 309.

In initial searches of public databases using the BLAST algorithm it wasfound that MLSK-1 most closely resembles (61.5% amino acid identity, 281of 452 residues) a putative human protein called KIAA0537 (GenBankaccession #AB011109), which also appears to encode a protein kinase.MLSK-1 is not likely to simply be the murine homolog of KIAA0537,however, since there is an example of a publicly-available human EST(AI469033) that shares a higher percentage amino acid identity (87%)with MLSK-1 over 477 predicted residues than does KIAA0537. Generally,MLSK-1 most closely resembles a subset of the protein kinase superfamilyknown as the adenosine monophosphate (AMP) kinases (AMPK's). More recentdatabase searches have not revealed any sequences more similar toMLSK-1.

As described in Example 8, we have expressed an active form of MLSK-1and shown that when over-expressed in COS cells it activates the MAPkinase signaling pathway as evidenced by the generation ofphosphorylated forms of ERK (Extracellularly Regulated Kinase), andtherefore is likely involved in a pathway regulating mitogenesis.Over-expression of MLSK-1 had no effect on the stress-activated kinasepathway, as it did not result in activation of either JNK nor p38kinases.

As described in Example 9, assays to determine whether MLSK-1 couldactivate the transcription factor AP-1 were performed. AP-1 is atranscription factor known to be involved in the JNK and p38 signalingpathways. MLSK-1 was co-transfected with an AP-1-luciferase constructinto COS-7 cells in a standard AP-1-luciferase reporter assay. Theoverexpression of MLSK-1 did not activate AP-1 using this assay system.

We obtained some preliminary data on MLSK-1 substrate specificity usinga PhosphoSpots™ assay, as described in Example 5. This is a membranewhich has coupled to it 20 different peptide sequences that are known tohave sites recognized by known kinases. MLSK-1 added phosphate residuesto a number of peptide substrates recognized by kinases, and inparticular those such as protein kinase C, p34 cdc2 kinase, and one ofthe p42/p44 MAP kinase substrates.

MLSK-2

In MLSK-2, nucleotides 121 to 1053 of SEQ ID NO:5 encode an open readingframe which ends with an in-frame stop codon. The predicted polypeptideof 311 amino acids of SEQ ID NO:12 contains a methionine at position 1from which translation is presumably initiated. The sequence C-terminalto this methionine contains little more than a canonical proteinserine/threonine kinase domain. There are currently no close homologs ofMLSK-2 in the protein sequence databases. There are twopublicly-available human ESTs (GenBank Accession numbers AA018361 andAI333117) that are highly similar to regions of MLSK-2 (88% identicalover 156 predicted residues, and 97% identical over 116 residues,respectively), implying a high degree of sequence conservation of thiskinase.

In more recent searches of the public databases using BLAST, thefollowing Genbank protein sequence was found to have sequence similarityto the MLSK-2 polypeptide: Accession #AL117482 hypothetical protein[Homo sapiens] (92% amino acid identity, 205 of 221 residues). Thesearch of the Derwent GeneSeq patent database using the BLAST algorithmrevealed: Accession #Y27057, Human protein kinase HKPM-6 (97% amino acididentity, 199 of 205 residues); and Accession #Y23755, Protein involvedin eliciting a signal in HH-PTC, human homologue of the Drosophila fusedgene (95% amino acid identity, 196 of 206 residues).

As with MLSK-1, we have expressed an active form of MLSK-2 and shownthat when over-expressed in COS cells it activates the MAP kinasesignaling pathway as evidenced by the generation of phosphorylated formsof ERK. Over-expression of MLSK-2 had no effect on the stress-activatedkinase pathway, i.e it did not activate either JNK nor p38 kinases. (SeeExample 8 below.)

As was done with MLSK-1, assays to determine whether MLSK-2 couldactivate the transcription factor AP-1 were performed. MLSK-2 wasco-transfected with an AP-1-luciferase construct into COS-7 cells in astandard AP-1-luciferase reporter assay. The overexpression of MLSK-2did not activate AP-1 using this assay system. (See Example 9 below.)

ss4694 and LNRK-1

A protein serine/threonine kinase was isolated from human dendriticcells, beginning with a novel protein serine/threonine kinase calledss4694 (SEQ ID NO:6); the nucleotide sequence of which codes for amethionine followed by an open reading frame of 261 amino acids (SEQ IDNO:13). Included in this open reading frame are conserved motifs foundin all protein kinases. The predicted amino acids 27 through 257 of SEQID NO:13 are identical to the N-terminal 230 amino acids of anincomplete protein kinase KIAA0551 (GenBank Accession #AB011123). Infact, ss4694 provides the missing 27 amino-acid N-terminal region ofKIAA051. This is significant because this N-terminal sequence cannot befound in any public database at this time. By combining the twosequences ss4694 and KIAA0551, a full-length transcript of 4.08 kb canbe predicted as shown in SEQ ID NO:7. The translation of this sequence,which we call LNRK-1 (Large NIK-Related Kinase-1), is shown as SEQID:14.

By using PCR primers based upon the sequence of ss4694, it wasdetermined by performing an RT-PCR assay (as described in Example 7below) that the kinase was expressed at relatively high levels in humanspleen. 5′ and 3′ flanking PCR primers (5′ primer:ATGGCGAGCGACTCCCCGGCTCGAA (SEQ ID NO:15); 3′ primer:CCAGTTCATCATGGAATTTCTGTTGAGGG (SEQ ID NO:16)) were then designed basedupon the predicted full-length sequence of LNRK-1 and were used toamplify a 4.08 kb cDNA from a Clontech Marathon-ready human spleen cDNAlibrary. Sequencing of this PCR product confirmed its identity with thesequence predicted for LNKR-1.

In initial searches of the public databases, the strongest homology inthe kinase domain of LNRK-1 is with murine NIK (GenBank Accession#U88984) and human HPK/GCK-like kinase HGK (GenBank Accession#AF096300). There are also close similarities to a number of othermembers of a growing family of kinases collectively termed theGC-kinases (Germinal Center Kinases) (Kyrakis, J M, 1999, Signaling bythe germinal center kinase family of protein kinases, J Biol Chem274(9): 5259-62). Generally these kinases are implicated as upstreamactivators of the Jun terminal kinases (JNK's) which arecharacteristically activated in response to a variety of cellularstresses.

In more recent searches of the public databases using BLAST, thefollowing Genbank protein sequence was found to have sequence similarityto the LNRK-1 polypeptide: Accession #AF172264, Traf2 and NCKinteracting kinase, splice variant 1 [Homo sapiens] (100% amino acididentity, 1360 of 1360 residues). The search of the Derwent GeneSeqpatent database using the BLAST algorithm revealed: Accession #Y55932,Human ZC2 (92% amino acid identity, 927 of 1002 residues). LNRK-1 isidentical to one of the eight splice variants identified for the Traf2and Nck interacting kinase “TNIK” of GenBank Accession #AF172264. TheZC2 protein of GeneSeq Accession #Y55932 appears to be identical or atleast related to another of these “TNIK” splice variants. TNIK isdescribed as a Novel Germinal Center Kinase Family Member That ActivatesThe JNK Pathway and Regulates The Cytoskeleton (Fu et al., 1999, J.Biol. Chem. 274 (43): 30729-30737; which is incorporated by referenceherein).

We have assayed the tissue-specific expression for LNRK-1 as reported inExample 7, and found roughly equivalent ubiquitous expression of it withslightly higher levels in PBL and slightly lower levels in kidney,skeletal muscle, and small intestine.

Fragments

The invention also provides polypeptides and fragments of the kinasedomain of polypeptides of the invention that retain a desired biologicalactivity. Particular embodiments are directed to polypeptide fragmentsthat retain the ability to bind a “binding partner” or native cognates,substrates, or counter-structure molecules. Such a fragment may be asoluble polypeptide. In another embodiment, the polypeptides andfragments advantageously include regions that are conserved in thekinase family.

Also provided herein are polypeptide fragments comprising at least 10,at least 20, or at least 30, contiguous amino acids of the sequence ofSEQ ID NOs:8-14. Fragments derived from different domains find use instudies of signal transduction, and in regulating cellular processesassociated with transduction of biological signals. Polypeptidefragments also may be employed as immunogens, in generating antibodies.

Variants

Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein.

Variants may exhibit amino acid sequences that are at least 80%identical. Also contemplated are embodiments in which a polypeptide orfragment comprises an amino acid sequence that is at least 90%identical, at least 95% identical, at least 98% identical, at least 99%identical, or at least 99.9% identical to the preferred polypeptide orfragment thereof. Percent identity may be determined by visualinspection and mathematical calculation. Alternatively, the percentidentity of two protein sequences can be determined by comparingsequence information using the GAP computer program, based on thealgorithm of Needleman and Wunsch (J. Mol. Bio. 48:443, 1970) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The preferred default parameters for the GAP program include:(1) a scoring matrix, blosum62, as described by Henikoff and Henikoff(Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) a gap weight of 12; (3)a gap length weight of 4; and (4) no penalty for end gaps. Otherprograms used by one skilled in the art of sequence comparison may alsobe used.

The variants of the invention include, for example, those that resultfrom alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1-5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

Other derivatives include covalent or aggregative conjugates of thepolypeptides with other proteins or polypeptides, such as by synthesisin recombinant culture as N-terminal or C-terminal fusions. Examples offusion proteins are discussed below in connection with oligomers.Further, fusion proteins can comprise peptides added to facilitatepurification and identification. Such peptides include, for example,poly-His or the antigenic identification peptides described in U.S. Pat.No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One suchpeptide is the FLAG® peptide which is highly antigenic and provides anepitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinant protein. Amurine hybridoma designated 4E11 produces a monoclonal antibody thatbinds the FLAG® peptide in the presence of certain divalent metalcations, as described in U.S. Pat. No. 5,011,912, hereby incorporated byreference. The 4E11 hybridoma cell line has been deposited with theAmerican Type Culture Collection under accession no. HB 9259. Monoclonalantibodies that bind the FLAG® peptide are available from Eastman KodakCo., Scientific Imaging Systems Division, New Haven, Conn.

Among the variant polypeptides provided herein are variants of nativepolypeptides that retain the native biological activity or thesubstantial equivalent thereof. One example is a variant that binds withessentially the same binding affinity as does the native form. Bindingaffinity can be measured by conventional procedures, e.g., as describedin U.S. Pat. No. 5,512,457 and as set forth below.

Variants include polypeptides that are substantially homologous to thenative form, but which have an amino acid sequence different from thatof the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

A given amino acid may be replaced, for example, by a residue havingsimilar physiochemical characteristics. Examples of such conservativesubstitutions include substitution of one aliphatic residue for another,such as Ile, Val, Leu, or Ala for one another; substitutions of onepolar residue for another, such as between Lys and Arg, Glu and Asp, orGln and Asn; or substitutions of one aromatic residue for another, suchas Phe, Trp, or Tyr for one another. Other conservative substitutions,e.g., involving substitutions of entire regions having similarhydrophobicity characteristics, are well known.

Similarly, the DNAs of the invention include variants that differ from anative DNA sequence because of one or more deletions, insertions orsubstitutions, but that encode a biologically active polypeptide.

The invention further includes polypeptides of the invention with orwithout associated native-pattern glycosylation. Polypeptides expressedin yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells)can be similar to or significantly different from a native polypeptidein molecular weight and glycosylation pattern, depending upon the choiceof expression system. Expression of polypeptides of the invention inbacterial expression systems, such as E. coli, provides non-glycosylatedmolecules. Further, a given preparation may include multipledifferentially glycosylated species of the protein. Glycosyl groups canbe removed through conventional methods, in particular those utilizingglycopeptidase. In general, glycosylated polypeptides of the inventioncan be incubated with a molar excess of glycopeptidase (BoehringerMannheim).

Correspondingly, similar DNA constructs that encode various additions orsubstitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences are encompassed by theinvention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate substitutions,additions, or deletions to the nucleotide sequence encoding thesetriplets will result in prevention of attachment of carbohydrateresidues at the Asn side chain. Alteration of a single nucleotide,chosen so that Asn is replaced by a different amino acid, for example,is sufficient to inactivate an N-glycosylation site. Alternatively, theSer or Thr can by replaced with another amino acid, such as Ala. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846, herebyincorporated by reference.

In another example of variants, sequences encoding Cys residues that arenot essential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges upon folding orrenaturation.

Other variants are prepared by modification of adjacent dibasic aminoacid residues, to enhance expression in yeast systems in which KEX2protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

Production of Polypeptides and Fragments Thereof

Expression, isolation and purification of the polypeptides and fragmentsof the invention may be accomplished by any suitable technique,including but not limited to the following:

Expression Systems

The present invention provides recombinant cloning and expressionvectors containing DNA, as well as host cell containing the recombinantvectors. Expression vectors comprising DNA may be used to prepare thepolypeptides or fragments of the invention encoded by the DNA. A methodfor producing polypeptides comprises culturing host cells transformedwith a recombinant expression vector encoding the polypeptide, underconditions that promote expression of the polypeptide, then recoveringthe expressed polypeptides from the culture. The skilled artisan willrecognize that the procedure for purifying the expressed polypeptideswill vary according to such factors as the type of host cells employed,and whether the polypeptide is membrane-bound or a soluble form that issecreted from the host cell.

Any suitable expression system may be employed. The vectors include aDNA encoding a polypeptide or fragment of the invention, operably linkedto suitable transcriptional or translational regulatory nucleotidesequences, such as those derived from a mammalian, microbial, viral, orinsect gene. Examples of regulatory sequences include transcriptionalpromoters, operators, or enhancers, an mRNA ribosomal binding site, andappropriate sequences which control transcription and translationinitiation and termination. Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the DNA sequence.Thus, a promoter nucleotide sequence is operably linked to a DNAsequence if the promoter nucleotide sequence controls the transcriptionof the DNA sequence. An origin of replication that confers the abilityto replicate in the desired host cells, and a selection gene by whichtransformants are identified, are generally incorporated into theexpression vector.

In addition, a sequence encoding an appropriate signal peptide (nativeor heterologous) can be incorporated into expression vectors. A DNAsequence for a signal peptide (secretory leader) may be fused in frameto the nucleic acid sequence of the invention so that the DNA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

The skilled artisan will also recognize that the position(s) at whichthe signal peptide is cleaved may differ from that predicted by computerprogram, and may vary according to such factors as the type of hostcells employed in expressing a recombinant polypeptide. A proteinpreparation may include a mixture of protein molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site.

Suitable host cells for expression of polypeptides include prokaryotes,yeast or higher eukaryotic cells. Mammalian or insect cells aregenerally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

Prokaryotic Systems

Prokaryotes include gram-negative or gram-positive organisms. Suitableprokaryotic host cells for transformation include, for example, E. coli,Bacillus subtilis, Salmonella typhimurium, and various other specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus. In aprokaryotic host cell, such as E. coli, a polypeptide may include anN-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. An appropriate promoter and a DNA sequence areinserted into the pBR322 vector. Other commercially available vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λP_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λP_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) andpPLc28 (resident in E. coli RR1, ATCC 53082).

Yeast Systems

Alternatively, the polypeptides may be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia or Kluyveromyces, may also be employed.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the polypeptide. The α-factor leader sequence is often insertedbetween the promoter sequence and the structural gene sequence. See,e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or Insect Systems

Mammalian or insect host cell culture systems also may be employed toexpress recombinant polypeptides. Bacculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Established cell lines of mammalianorigin also may be employed. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al. (EMBO J. 10: 2821, 1991).

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipidreagent, can be used to transfect cells (Felgner et al., Proc. Natl.Acad. Sci. USA 84:7413-7417, 1987). In addition, electroporation can beused to transfect mammalian cells using conventional procedures, such asthose in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed.Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989). Selection ofstable transformants can be performed using methods known in the art,such as, for example, resistance to cytotoxic drugs. Kaufman et al.,Meth. in Enzymology 185:487-511, 1990, describes several selectionschemes, such as dihydrofolate reductase (DHFR) resistance. A suitablehost strain for DHFR selection can be CHO strain DX-B11, which isdeficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA77:4216-4220, 1980). A plasmid expressing the DHFR cDNA can beintroduced into strain DX-B11, and only cells that contain the plasmidcan grow in the appropriate selective media. Other examples ofselectable markers that can be incorporated into an expression vectorinclude cDNAs conferring resistance to antibiotics, such as G418 andhygromycin B. Cells harboring the vector can be selected on the basis ofresistance to these compounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., Animal Cell Technology, 1997, pp. 529-534 and PCTApplication WO 97/25420) and the tripartite leader (TPL) and VA geneRNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475-13491,1982). The internal ribosome entry site (IRES) sequences of viral originallows dicistronic mRNAs to be translated efficiently (Oh and Sarnow,Current Opinion in Genetics and Development 3:295-300, 1993; Ramesh etal., Nucleic Acids Research 24:2697-2700, 1996). Expression of aheterologous cDNA as part of a dicistronic mRNA followed by the gene fora selectable marker (e.g. DHFR) has been shown to improvetransfectability of the host and expression of the heterologous cDNA(Kaufman, Meth. in Enzymology, 1990). Exemplary expression vectors thatemploy dicistronic mRNAs are pTR-DC/GFP described by Mosser et al.,Biotechniques 22:150-161, 1997, and p2A5I described by Morris et al.,Animal Cell Technology, 1997, pp. 529-534.

A useful high expression vector, pCAVNOT, has been described by Mosleyet al., Cell 59:335-348, 1989. Other expression vectors for use inmammalian host cells can be constructed as disclosed by Okayama and Berg(Mol. Cell. Biol. 3:280, 1983). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. (Mol.Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4,described by Cosman et al., Nature 312:768, 1984, has been deposited asATCC 39890. Additional useful mammalian expression vectors are describedin EP-A-0367566, and in WO 91/18982, incorporated by reference herein.In yet another alternative, the vectors can be derived fromretroviruses.

Additional useful expression vectors, pFLAG® and pDC311, can also beused. FLAG® technology is centered on the fusion of a low molecularweight (1 kD), hydrophilic, FLAG® marker peptide to the N-terminus of arecombinant protein expressed by pFLAG® expression vectors. pDC311 isanother specialized vector used for expressing proteins in CHO cells.pDC311 is characterized by a bicistronic sequence containing the gene ofinterest and a dihydrofolate reductase (DHFR) gene with an internalribosome binding site for DHFR translation, an expression augmentingsequence element (EASE), the human CMV promoter, a tripartite leadersequence, and a polyadenylation site.

Regarding signal peptides that may be employed, the native signalpeptide may be replaced by a heterologous signal peptide or leadersequence, if desired. The choice of signal peptide or leader may dependon factors such as the type of host cells in which the recombinantpolypeptide is to be produced. To illustrate, examples of heterologoussignal peptides that are functional in mammalian host cells include thesignal sequence for interleukin-7 (IL-7) described in U.S. Pat.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312:768 (1984); the interleukin-4 receptor signalpeptide described in EP 367,566; the type I interleukin-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846.

Purification

The invention also includes methods of isolating and purifying thepolypeptides and fragments thereof.

Isolation and Purification

The “isolated” polypeptides or fragments thereof encompassed by thisinvention are polypeptides or fragments that are not in an environmentidentical to an environment in which it or they can be found in nature.The “purified” polypeptides or fragments thereof encompassed by thisinvention are essentially free of association with other proteins orpolypeptides, for example, as a purification product of recombinantexpression systems such as those described above or as a purifiedproduct from a non-recombinant source such as naturally occurring cellsand/or tissues.

In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention. Suchfusion partners can include the poly-His or other antigenicidentification peptides described above as well as the Fc moietiesdescribed previously.

With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant polypeptide or fragmentwill vary according to such factors as the type of host cells employedand whether or not the recombinant polypeptide or fragment is secretedinto the culture medium.

In general, the recombinant polypeptide or fragment can be isolated fromthe host cells if not secreted, or from the medium or supernatant ifsoluble and secreted, followed by one or more concentration,salting-out, ion exchange, hydrophobic interaction, affinitypurification or size exclusion chromatography steps. As to specific waysto accomplish these steps, the culture medium first can be concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. In addition, a chromatofocusingstep can be employed. Alternatively, a hydrophobic interactionchromatography step can be employed. Suitable matrices can be phenyl oroctyl moieties bound to resins. In addition, affinity chromatographywith a matrix which selectively binds the recombinant protein can beemployed. Examples of such resins employed are lectin columns, dyecolumns, and metal-chelating columns. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel or polymer resinhaving pendant methyl, octyl, octyldecyl or other aliphatic groups) canbe employed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantprotein.

It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention.

In this aspect of the invention, polypeptide-binding proteins, such asthe anti-polypeptide antibodies of the invention or other proteins thatmay interact with the polypeptide of the invention, can be bound to asolid phase support such as a column chromatography matrix or a similarsubstrate suitable for identifying, separating, or purifying cells thatexpress polypeptides of the invention on their surface. Adherence ofpolypeptide-binding proteins of the invention to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See Berenson, et al. J. Cell. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

The desired degree of purity depends on the intended use of the protein.A relatively high degree of purity is desired when the polypeptide is tobe administered in vivo, for example. In such a case, the polypeptidesare purified such that no protein bands corresponding to other proteinsare detectable upon analysis by SDS-polyacrylamide gel electrophoresis(SDS-PAGE). It will be recognized by one skilled in the pertinent fieldthat multiple bands corresponding to the polypeptide may be visualizedby SDS-PAGE, due to differential glycosylation, differentialpost-translational processing, and the like. Most preferably, thepolypeptide of the invention is purified to substantial homogeneity, asindicated by a single protein band upon analysis by SDS-PAGE. Theprotein band may be visualized by silver staining, Coomassie bluestaining, or (if the protein is radiolabeled) by autoradiography.

Use of Nucleic Acid or Oligonucleotides of the Invention

In addition to being used to express polypeptides as described above,the nucleic acids of the invention, including DNA, RNA, mRNA andoligonucleotides thereof can be used as probes to study signaltransduction and to identify nucleic acid encoding proteins havingkinase activity; as diagnostic disease markers; and as single-strandedsense or antisense oligonucleotides to inhibit expression of polypeptideencoded by the genes of the invention.

Probes

Among the uses of nucleic acids of the invention is the use of fragmentsas probes or primers. Such fragments generally comprise at least about17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNAfragment comprises at least 30, or at least 60, contiguous nucleotidesof a DNA sequence.

Because homologs of SEQ ID NOs:1-7 from other mammalian species arecontemplated herein, probes based on the DNA sequence of SEQ ID NOs:1-7may be used to screen cDNA libraries derived from other mammalianspecies, using conventional cross-species hybridization techniques.

Using knowledge of the genetic code in combination with the amino acidsequences set forth above, sets of degenerate oligonucleotides can beprepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified.

Chromosome Mapping

All or a portion of the nucleic acids of SEQ ID NO:1-7, includingoligonucleotides, can be used by those skilled in the art usingwell-known techniques to identify the human chromosome, and the specificlocus thereof. Useful techniques include, but are not limited to, usingthe sequence or portions, including oligonucleotides, as a probe invarious well-known techniques such as radiation hybrid mapping (highresolution), in situ hybridization to chromosome spreads (moderateresolution), and Southern blot hybridization to hybrid cell linescontaining individual human chromosomes (low resolution).

For example, chromosomes can be mapped by radiation hybridization.First, PCR is performed using the Whitehead Institute/MYI Center forGenome Research Genebridge4 panel of 93 radiation hybrids(www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/rhmap/genebridge4.html).Primers are used which lie within a putative exon of the gene ofinterest and which amplify a product from human genomic DNA, but do notamplify hamster genomic DNA. The results of the PCRs are converted intoa data vector that is submitted to the Whitehead/MIT Radiation Mappingsite on the internet (http://www-seq.wi.mit.edu). The data is scored andthe chromosomal assignment and placement relative to known Sequence TagSite (STS) markers on the radiation hybrid map is provided. Thefollowing web site provides additional information about radiationhybrid mapping:http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/07-97.INTRO.html).

Sense-Antisense

Other useful fragments of the nucleic acids include antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences. Antisense or sense oligonucleotides, according tothe present invention, comprise a fragment of DNA (SEQ ID NOs:1-7). Sucha fragment generally comprises at least about 14 nucleotides, preferablyfrom about 14 to about 30 nucleotides. The ability to derive anantisense or a sense oligonucleotide, based upon a cDNA sequenceencoding a given protein is described in, for example, Stein and Cohen(Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block or inhibitprotein expression by one of several means, including enhanceddegradation of the mRNA by RNAseH, inhibition of splicing, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression ofproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10448, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, lipofection, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus.

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Use of Kinase Polypeptides and Fragmented Polypeptides

Uses include, but are not limited to, the following:

Identifying molecules that modulate kinase activity and cellularresponses

Purifying proteins and measuring activity thereof

Research Reagents

Molecular weight and Isoelectric focusing markers

Controls for peptide fragmentation

Identification of unknown proteins

Preparation of Antibodies

Therapeutic Compounds

Identifying molecules that modulate kinase activity and cellularresponses

Cellular signaling often involves a molecular activation cascade, duringwhich a receptor propagates a ligand-receptor mediated signal byspecifically activating intracellular kinases which phosphorylate targetsubstrates. These substrates can themselves be kinases which becomeactivated following phosphorylation. Alternatively, they can be adaptormolecules that facilitate down stream signaling through protein-proteininteraction following phosphorylation. Regardless of the nature of thesubstrate molecule(s), expressed functionally active versions of thepolypeptides of the invention, for example the kinase domain, can beused in assays to identify molecules that modulate the recognition andactivation of substrate(s) by the kinases. These assays include, withoutlimitation, the yeast 1,- 2-, or 3-hybrid assays, or an assay of bindingthat measures changes in the surface plasmon resonance of a boundmolecule (using for example an instrument from BIAcore), or other assaysdesigned for high-throughut screening. Other assays that may be used toidentify activators and inhibitors of the kinases of the invention aredescribed without limitation in the Examples below. In this way, thesenovel kinases can be used in assay methods to identify novel moleculesthat can modulate the activity of signal transduction pathways andaffect the cellular response to external and internal signals.

The purpose of such an assay is to identify substances which modulatesubstrate phosphorylation. Such inhibitory or activating substancescould serve as lead compounds in the development of pharmaceuticals forthe treatment of, for example, autoimmune disease, rheumatoid and otherinflammatory conditions, cancer, viral infections, asthma, transplantrejection, infectious or neoplastic diseases in which there is adisregulation of processes mediated by the kinase. It is conceivablethat compounds which activate or inhibit the kinases of the inventioncould have merit as more general modulators of the class of proteinkinases which mediate signaling, including (but not limited to) thosementioned herein. Examples of compounds that may modulate kinaseactivity include but are not limited to catalytically inactive,truncated, or otherwise mutated form of a kinase, preferably a kinase ofthe present invention; and numerous chemical classes, particularlyorganic compounds; preferably small organic compounds and are obtainedfrom a wide variety of sources including libraries of synthetic ornatural compounds. Preferred modulators of kinase activity are orallyactive in mammalian hosts. For diagnostic uses, the kinase modulators orother binding agents are frequently labeled, such as with fluorescent,radioactive, chemiluminescent, or other easily detectable molecules,either conjugated directly to the binding agent or conjugated to a probespecific for the binding agent.

Purification Reagents

Each of the polypeptides of the invention finds use as a proteinpurification reagent. For example, the polypeptides may be used topurify binding partner proteins. In particular embodiments, apolypeptide (in any form described herein that is capable of binding abinding partner) is attached to a solid support by conventionalprocedures. As one example, affinity chromatography columns containingfunctional groups that will react with functional groups on amino acidside chains of proteins are available (Pharmacia Biotech, Inc.,Piscataway, N.J.).

The polypeptide also finds use in identifying cells that express bindingpartner proteins. Polypeptides are bound to a solid phase such as acolumn chromatography matrix or a similar suitable substrate. Forexample, magnetic microspheres can be coated with the polypeptides andheld in an incubation vessel through a magnetic field. Suspensions ofcell lysates containing expressed binding partner protein are contactedwith the solid phase having the polypeptides thereon. Binding partnerproteins from cells expressing the binding partner bind to the fixedpolypeptides, and unbound protein is washed away.

Alternatively, the polypeptides can be conjugated to a detectablemoiety, then incubated with cell lysates to be tested for bindingpartner expression. After incubation, unbound labeled matter is removedand the presence or absence of the detectable moiety is determined.

In a further alternative, mixtures of cell lysates suspected ofcontaining binding partner protein are incubated with biotinylatedpolypeptides of the invention. Incubation periods are typically at leastone hour in duration to ensure sufficient binding. The resulting mixturethen is passed through a column packed with avidin-coated beads, wherebythe high affinity of biotin for avidin provides binding of the desiredcells to the beads. Procedures for using avidin-coated beads are known(see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing toremove unbound material, and the release of the bound cells, areperformed using conventional methods.

Measuring Activity

Polypeptides also find use in measuring the biological activity ofbinding partner protein in terms of their binding affinity. Thepolypeptides thus may be employed by those conducting “qualityassurance” studies, e.g., to monitor shelf life and stability of proteinunder different conditions. For example, the polypeptides may beemployed in a binding affinity study to measure the biological activityof a binding partner protein that has been stored at differenttemperatures, or produced in different cell types. The proteins also maybe used to determine whether biological activity is retained aftermodification of a binding partner protein (e.g., chemical modification,truncation, mutation, etc.). The binding affinity of the modifiedbinding partner protein is compared to that of an unmodified bindingpartner protein to detect any adverse impact of the modifications onbiological activity of binding partner. The biological activity of abinding partner protein thus can be ascertained before it is used in aresearch study, for example.

In particularly preferred embodiments, the isolated kinase polypeptidefragments can be used to assay protein kinase activity.

Research Agents

Another embodiment of the invention is the use of isolated kinasepolypeptides of the invention, fusion proteins, or a fragment thereofcontaining the isolated protein kinase domain in in vitro or in vivoassays to determine protein kinase activity. A hallmark of proteinkinases is their ability to phosphorylate other proteins and toauto-phosphorylate. Therefore, in one aspect of the invention, theisolated polypeptides with kinase activity can be used in assays tophosphorylate target proteins, radiolabel target proteins with ³²P, andidentify proteins having phosphatase activity. Exemplary methods ofphosphorylation assays set forth above are disclosed in U.S. Pat. No.5,447,860 which is incorporated herein by reference. In addition to fulllength polypeptides, the invention also includes the isolated activekinase domains of kinases which can function as reagents in kinaseassays.

Kinase assays are typically carried out by combining a kinase of theinvention, or an active kinase domain, with radiolabeled ATP (γ³²P-ATP)and a peptide or protein substrate in a buffer solution. The peptidesubstrates generally range from 8 to 30 amino acids in length or thesubstrate may also be a protein known to be phosphorylated readily by akinase of the invention. Many such general kinase substrates are known,e.g. α or β casein, histone H1, myelin basic protein, etc. Afterincubation of this reaction mixture at 20-37° C. for a suitable time,the kinase mediated transfer of radioactive phosphate from ATP to thesubstrate protein or substrate peptide can be determined by methods wellknown in the art, such as, for example, spotting the radioactiveproducts onto phosphocellulose paper, followed by washing and liquidscintillation counting, gel electrophoresis followed by autoradiography,and scintillation proximity assay.

Another embodiment of the invention relates to the study of cell signaltransduction. The kinases of the invention, like other kinases, couldplay a central role in immune responses or other cellular process whichinclude cellular signal transduction. As such, alterations in theexpression and/or activation of the kinases can have profound effects ona plethora of cellular processes. Expression of cloned kinases,functionally inactive mutants thereof, or the kinase domain can be usedto identify the role a particular protein plays in mediating specificsignaling events.

Cellular signaling often involves a molecular activation cascade, duringwhich a receptor propagates a ligand-receptor mediated signal byspecifically activating intracellular kinases which phosphorylate targetsubstrates. These substrates can themselves be kinases which becomeactivated following phosphorylation. Alternatively, they can be adaptormolecules that facilitate down stream signaling through protein-proteininteraction following phosphorylation. Regardless of the nature of thesubstrate molecule(s), expressed functionally active versions of thepolypeptides of the invention, for example the kinase domain, can beused in assays such as the yeast 2-hybrid assay to identify whatsubstrate(s) were recognized and activated by the kinase bindingpartner(s). As such, these novel kinases can be used as reagents toidentify novel molecules involved in signal transduction pathways.

More specifically, MDCK-1 and LNRK-1, with their strong homologies tohuman HPK/GCK-like kinase HGK and murine NIK and similarities to othermembers of the GC (for Germinal Center) kinase family, may be implicatedas upstream activators of the Jun terminal kinases. Because JNKs arecharacteristically activated in response to a variety of cellularsignals, MDCK-1 and LNRK-1 (as cDNA, polypeptides, or antibodies) may beused for the study of signal transduction cascades in tissues where thekinases are expressed.

Molecular Weight, Isoelectric Point Markers

The polypeptides of the present invention can be subjected tofragmentation into smaller peptides by chemical and enzymatic means, andthe peptide fragments so produced can be used in the analysis of otherproteins or polypeptides. For example, such peptide fragments can beused as peptide molecular weight markers, peptide isoelectric pointmarkers, or in the analysis of the degree of peptide fragmentation.Thus, the invention also includes these polypeptides and peptidefragments, as well as kits to aid in the determination of the apparentmolecular weight and isoelectric point of an unknown protein and kits toassess the degree of fragmentation of an unknown protein.

Although all methods of fragmentation are encompassed by the invention,chemical fragmentation is a preferred embodiment, and includes the useof cyanogen bromide to cleave under neutral or acidic conditions suchthat specific cleavage occurs at methionine residues (E. Gross, Methodsin Enz. 11:238-255, 1967). This can further include additional steps,such as a carboxymethylation step to convert cysteine residues to anunreactive species.

Enzymatic fragmentation is another preferred embodiment, and includesthe use of a protease such as Asparaginylendo-peptidase,Arginylendo-peptidase, Achromobacter protease I, Trypsin, Staphlococcusaureus V8 protease, Endoproteinase Asp-N, or Endoproteinase Lys-C underconventional conditions to result in cleavage at specific amino acidresidues. Asparaginylendo-peptidase can cleave specifically on thecarboxyl side of the asparagine residues present within the polypeptidesof the invention. Arginylendo-peptidase can cleave specifically on thecarboxyl side of the arginine residues present within thesepolypeptides. Achromobacter protease I can cleave specifically on thecarboxyl side of the lysine residues present within the polypeptides(Sakiyama and Nakat, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim.Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim. Biophys. Acta660:51-55, 1981). Trypsin can cleave specifically on the carboxyl sideof the arginine and lysine residues present within polypeptides of theinvention. Enzymatic fragmentation may also occur with a protease thatcleaves at multiple amino acid residues. For example, Staphlococcusaureus V8 protease can cleave specifically on the carboxyl side of theaspartic and glutamic acid residues present within polypeptides (D. W.Cleveland, J. Biol. Chem. 3:1102-1106, 1977). Endoproteinase Asp-N cancleave specifically on the amino side of the asparagine residues presentwithin polypeptides. Endoproteinase Lys-C can cleave specifically on thecarboxyl side of the lysine residues present within polypeptides of theinvention. Other enzymatic and chemical treatments can likewise be usedto specifically fragment these polypeptides into a unique set ofspecific peptides.

Of course, the peptides and fragments of the polypeptides of theinvention can also be produced by conventional recombinant processes andsynthetic processes well known in the art. With regard to recombinantprocesses, the polypeptides and peptide fragments encompassed byinvention can have variable molecular weights, depending upon the hostcell in which they are expressed.

The molecular weight of these polypeptides can also be varied by fusingadditional peptide sequences to both the amino and carboxyl terminalends of polypeptides of the invention. Fusions of additional peptidesequences at the amino and carboxyl terminal ends of polypeptides of theinvention can be used to enhance expression of these polypeptides or aidin the purification of the protein. In addition, fusions of additionalpeptide sequences at the amino and carboxyl terminal ends ofpolypeptides of the invention will alter some, but usually not all, ofthe fragmented peptides of the polypeptides generated by enzymatic orchemical treatment. Of course, mutations can be introduced intopolypeptides of the invention using routine and known techniques ofmolecular biology. For example, a mutation can be designed so as toeliminate a site of proteolytic cleavage by a specific enzyme or a siteof cleavage by a specific chemically induced fragmentation procedure.The elimination of the site will alter the peptide fingerprint ofpolypeptides of the invention upon fragmentation with the specificenzyme or chemical procedure.

Because the unique amino acid sequence of each fragment specifies amolecular weight, these fragments can thereafter serve as molecularweight markers using such analysis techniques to assist in thedetermination of the molecular weight of an unknown protein,polypeptides or fragments thereof. The molecular weight markers of theinvention serve particularly well as molecular weight markers for theestimation of the apparent molecular weight of proteins that havesimilar apparent molecular weights and, consequently, allow increasedaccuracy in the determination of apparent molecular weight of proteins.

When the invention relates to the use of fragmented peptide molecularweight markers, those markers are preferably at least 10 amino acids insize. More preferably, these fragmented peptide molecular weight markersare between 10 and 100 amino acids in size. Even more preferable arefragmented peptide molecular weight markers between 10 and 50 aminoacids in size and especially between 10 and 35 amino acids in size. Mostpreferable are fragmented peptide molecular weight markers between 10and 20 amino acids in size.

Among the methods for determining molecular weight are sedimentation,gel electrophoresis, chromatography, and mass spectrometry. Aparticularly preferred embodiment is denaturing polyacrylamide gelelectrophoresis (U. K. Laemmli, Nature 227:680-685, 1970).Conventionally, the method uses two separate lanes of a gel containingsodium dodecyl sulfate and a concentration of acrylamide between 6-20%.The ability to simultaneously resolve the marker and the sample underidentical conditions allows for increased accuracy. It is understood, ofcourse, that many different techniques can be used for the determinationof the molecular weight of an unknown protein using polypeptides of theinvention, and that this embodiment in no way limits the scope of theinvention.

Each unglycosylated polypeptide or fragment thereof has a pI that isintrinsically determined by its unique amino acid sequence (which pI canbe estimated by the skilled artisan using any of the computer programsdesigned to predict pI values currently available, calculated using anywell-known amino acid pKa table, or measured empirically). Thereforethese polypeptides and fragments thereof can serve as specific markersto assist in the determination of the isoelectric point of an unknownprotein, polypeptide, or fragmented peptide using techniques such asisoelectric focusing. These polypeptide or fragmented peptide markersserve particularly well for the estimation of apparent isoelectricpoints of unknown proteins that have apparent isoelectric points closeto that of the polypeptide or fragmented peptide markers of theinvention.

The technique of isoelectric focusing can be further combined with othertechniques such as gel electrophoresis to simultaneously separate aprotein on the basis of molecular weight and charge. The ability tosimultaneously resolve these polypeptide or fragmented peptide markersand the unknown protein under identical conditions allows for increasedaccuracy in the determination of the apparent isoelectric point of theunknown protein. This is of particular interest in techniques, such astwo dimensional electrophoresis (T. D. Brock and M. T. Madigan, Biologyof Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)), where the natureof the procedure dictates that any markers should be resolvedsimultaneously with the unknown protein. In addition, with such methods,these polypeptides and fragmented peptides thereof can assist in thedetermination of both the isoelectric point and molecular weight of anunknown protein or fragmented peptide.

Polypeptides and fragmented peptides can be visualized using twodifferent methods that allow a discrimination between the unknownprotein and the molecular weight markers. In one embodiment, thepolypeptide and fragmented peptide molecular weight markers of theinvention can be visualized using antibodies generated against thesemarkers and conventional immunoblotting techniques. This detection isperformed under conventional conditions that do not result in thedetection of the unknown protein. It is understood that it may not bepossible to generate antibodies against all polypeptide fragments of theinvention, since small peptides may not contain immunogenic epitopes. Itis further understood that not all antibodies will work in this assay;however, those antibodies which are able to bind polypeptides andfragments of the invention can be readily determined using conventionaltechniques.

The unknown protein is also visualized by using a conventional stainingprocedure. The molar excess of unknown protein to polypeptide orfragmented peptide molecular weight markers of the invention is suchthat the conventional staining procedure predominantly detects theunknown protein. The level of these polypeptide or fragmented peptidemolecular weight markers is such as to allow little or no detection ofthese markers by the conventional staining method. The preferred molarexcess of unknown protein to polypeptide molecular weight markers of theinvention is between 2 and 100,000 fold. More preferably, the preferredmolar excess of unknown protein to these polypeptide molecular weightmarkers is between 10 and 10,000 fold and especially between 100 and1,000 fold.

It is understood of course that many techniques can be used for thedetermination and detection of molecular weight and isoelectric point ofan unknown protein, polypeptides, and fragmented peptides thereof usingthese polypeptide molecular weight markers and peptide fragments thereofand that these embodiments in no way limit the scope of the invention.

In another embodiment, the analysis of the progressive fragmentation ofthe polypeptides of the invention into specific peptides (D. W.Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977), such as byaltering the time or temperature of the fragmentation reaction, can beused as a control for the extent of cleavage of an unknown protein. Forexample, cleavage of the same amount of polypeptide and unknown proteinunder identical conditions can allow for a direct comparison of theextent of fragmentation. Conditions that result in the completefragmentation of the polypeptide can also result in completefragmentation of the unknown protein.

As to the specific use of the polypeptides and fragmented peptides ofthe invention as molecular weight markers, the fragmentation of thepolypeptide of SEQ ID NOs:8-14 with cyanogen bromide generates a uniqueset of fragmented peptide molecular weight markers (See Tables I-VI).The distribution of methionine residues determines the number of aminoacids in each peptide and the unique amino acid composition of eachpeptide determines its molecular weight.

TABLE I Cleavage of MDCK-1 with cyanogen bromide Position From-ToMolecular Weight 1 1-1 149.2 8 237-239 383.5 7 227-236 1003.1 3 57-721951.0 6 196-226 3459.7 4  73-105 3892.5 2  2-56 5810.6 9 240-313 8251.65 106-195 10037.5

TABLE II Cleavage of MDCK-2 with cyanogen bromide Position From-ToMolecular Weight 1 1-1 149.2 12 296-303 671.7 7 153-158 784.9 2 2-8793.8 10 230-241 1327.5 3  9-26 2038.4 9 204-229 3102.5 6 123-152 3744.58 159-203 4838.6 4 27-71 5089.9 5  72-122 5790.5 11 242-295 6385.2

TABLE III Cleavage of MDCK-3 with cyanogen bromide Position From-ToMolecular Weight 1 1-1 149.2 6 172-178 734.8 5 163-171 997.2 4 139-1622878.2 9 320-355 3709.0 3  99-138 4662.3 8 261-319 6794.6 7 179-2609284.3 2  2-98 10881.4

TABLE IV Cleavage of MLSK-1 with cyanogen bromide Position From-ToMolecular Weight 1 1-1 149.2 4 109-133 2815.3 2  2-39 3888.4 6 253-2894182.8 7 290-336 5186.6 8 337-387 5736.5 3  40-108 8283.6 5 134-25213554.3 9 388-631 26152.9

TABLE V Cleavage of MLSK-2 with cyanogen bromide Position From-ToMolecular Weight 1 1-1 149.2 6 181-184 446.5 5 165-180 1915.2 3  92-1183149.7 8 265-311 5276.8 4 119-164 5279.1 7 185-264 9036.3 2  2-9110174.7

TABLE VI Cleavage of LNRK-1 with cyanogen bromide Position From-ToMolecular Weight 11 285-431 18345.0 17 824-879 5992.1 12 432-525 12060.53 57-72 1879.0 13 526-551 2971.3 25 1253-1254 278.3 20 958-965 858.9 11-1 149.2 31 1358-1358 149.2 30 1351-1357 834.9 19 905-957 5627.1 9237-239 383.5 32 1359-1360 318.3 18 880-904 2576.8 8 227-236 1003.1 221070-1091 2693.1 5  98-105 1019.2 27 1283-1295 1460.7 28 1296-13172431.7 29 1318-1350 3877.5 26 1255-1282 3280.7 14 552-584 3584.0 4 73-972935.4 15 585-635 5487.2 7 196-226 3415.7 16 636-823 20422.3 2  2-565858.6 10 240-284 5311.2 21  966-1069 11599.8 6 106-195 10251.7 241181-1252 7943.0 23 1092-1180 10501.4

In addition, the preferred purified polypeptide of the invention (SEQ IDNOs:8-14) have calculated molecular weights in the absence ofglycosylation as follows:

TABLE VII Polypeptide Daltons MDCK-1 (SEQ ID NO:8) M_(r)˜34,813 andMDCK-2 (SEQ ID NO:9) M_(r)˜34,537 (from MET 3) M_(r)˜33,761 (from MET10) M_(r)˜31,740 (from MET 28) MDCK-3 (SEQ ID NO:10) M_(r)˜39,947 MLSK-1(SEQ ID NO:11) M_(r)˜69,806 MLSK-2 (SEQ ID NO:13) M_(r)˜39,477 SS4694(SEQ ID NO:13) M_(r)˜29,954 LNRK-1 (SEQ ID NO:14) M_(r)˜154,943

Finally, as to the kits that are encompassed by the invention, theconstituents of such kits can be varied, but typically contain thepolypeptide and fragmented peptide molecular weight markers. Also, suchkits can contain the polypeptides wherein a site necessary forfragmentation has been removed. Furthermore, the kits can containreagents for the specific cleavage of the polypeptide and the unknownprotein by chemical or enzymatic cleavage. Kits can further containantibodies directed against polypeptides or fragments thereof of theinvention.

Identification of Unknown Proteins

As set forth above, a polypeptide or peptide fingerprint can be enteredinto or compared to a database of known proteins to assist in theidentification of the unknown protein using mass spectrometry (W. J.Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; D. Fenyoet al., Electrophoresis 19:998-1005, 1998). A variety of computersoftware programs to facilitate these comparisons are accessible via theInternet, such as Protein Prospector (Internet site:prospector.uscf.edu), MultiIdent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internetsite:www.mann.emblheiedelberg.de...deSearch/FR_PeptideSearch Form.html),and ProFound (Internet site: www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programs allow the user to specify thecleavage agent and the molecular weights of the fragmented peptideswithin a designated tolerance. The programs compare observed molecularweights to predicted peptide molecular weights derived from sequencedatabases to assist in determining the identity of the unknown protein.

In addition, a polypeptide or peptide digest can be sequenced usingtandem mass spectrometry (MS/MS) and the resulting sequence searchedagainst databases (J. K. Eng, et al., J. Am. Soc. Mass Spec. 5:976-989(1994); M. Mann and M. Wilm, Anal. Chem. 66:43904399 (1994); J. A.Taylor and R. S. Johnson, Rapid Comm. Mass Spec.11: 1067-1075 (1997)).Searching programs that can be used in this process exist on theInternet, such as Lutefisk 97 (Internet site:www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, PeptideSearch and ProFound programs described above.

Therefore, adding the sequence of a gene and its predicted proteinsequence and peptide fragments to a sequence database can aid in theidentification of unknown proteins using mass spectrometry.

Antibodies

Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

Thus, one aspect of the present invention relates to the antigenicepitopes of the polypeptides of the invention. Such epitopes are usefulfor raising antibodies, in particular monoclonal antibodies, asdescribed in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Hybridoma cell lines that produce monoclonal antibodies specific for thepolypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies may be prepared by known techniques and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

Antigen-binding fragments of the antibodies, which may be produced byconventional techniques, are also encompassed by the present invention.Examples of such fragments include, but are not limited to, Fab andF(ab′)₂ fragments. Antibody fragments and derivatives produced bygenetic engineering techniques are also provided.

In one embodiment, the antibodies are specific for the polypeptides ofthe present invention and do not cross-react with other proteins.Screening procedures by which such antibodies may be identified are wellknown, and may involve immunoaffinity chromatography, for example.

Uses Thereof

The antibodies of the invention can be used in assays to detect thepresence of the polypeptides or fragments of the invention, either invitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography.

Those antibodies that additionally can block binding of the polypeptidesof the invention to a binding partner may be used to inhibit abiological activity that results from such binding. Such blockingantibodies may be identified using any suitable assay procedure, such asby testing antibodies for the ability to inhibit binding of kinases ofthe invention to the binding partner. Alternatively, blocking antibodiesmay be identified in assays for the ability to inhibit a biologicaleffect that results from binding of kinases to binding partners.

Such an antibody may be employed in an in vitro procedure, oradministered in vivo to inhibit a biological activity mediated by theentity that generated the antibody. Disorders caused or exacerbated(directly or indirectly) by the interaction of kinases with bindingpartners thus may be treated. A therapeutic method involves in vivoadministration of a blocking antibody to a mammal in an amount effectivein inhibiting a binding partner-mediated biological activity. Monoclonalantibodies are generally preferred for use in such therapeutic methods.In one embodiment, an antigen-binding antibody fragment is employed.

Antibodies may be screened for agonistic (i.e., ligand-mimicking)properties. Such antibodies, upon binding to a binding partner, inducebiological effects (e.g., transduction of biological signals) similar tothe biological effects induced when the kinases of the invention bind tobinding partners.

Compositions comprising an anti-kinase antibody, and a physiologicallyacceptable diluent, excipient, or carrier, are provided herein. Suitablecomponents of such compositions are as described above for compositionscontaining kinase proteins.

Also provided herein are conjugates comprising a detectable (e.g.,diagnostic) or therapeutic agent, attached to the antibody. Examples ofsuch agents are presented above. The conjugates find use in in vitro orin vivo procedures.

Therapeutic Activities

The polynucleotides and proteins of the present invention are expectedto exhibit one or more of the therapuetic uses or biological activities(including those associated with assays cited herein) identified below.Therapeutic uses or activities described for proteins of the presentinvention may be provided by administration or use of such proteins orby administration or use of polynucleotides encoding such proteins (suchas, for example, in gene therapies or vectors suitable for introductionof DNA).

Cytokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit cytokine-inducing, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce or inhibitproduction of cytokines in certain cell populations. Thecell-proliferation activity of a protein of the present invention isevidenced by any one of a number of routine factor dependent cellproliferation assays for cell lines including, without limitation, 32D,DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123,T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of a protein of theinvention may, among other means, be measured by the following methods:Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J.Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761,1994. Assays for cytokine production and/or proliferation of spleencells, lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human Interferon gamma., Schreiber, R. D. InCurrent Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994. Assays forproliferation and differentiation of hematopoietic and lymphopoieticcells include, without limitation, those described in: Measurement ofHuman and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis,L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a.Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991;deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan,R. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc.Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of humanInterleukin 11-Bennett, F., Giannotti, J., Clark, S. C. and Turner, K.J. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse andhuman Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. andTurner, K. J. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Immune Stimulating or Suppressing Activity

A protein of the present invention may also exhibit immune stimulatingor immune suppressing activity, including without limitation theactivities for which assays are described herein. A protein may beuseful in the treatment of various immune deficiencies and disorders(including severe combined immunodeficiency (SCID)), e.g., in regulating(up or down) growth and proliferation of T and/or B lymphocytes, as wellas effecting the cytolytic activity of NK cells and other cellpopulations. These immune deficiencies may be genetic or be caused byviral (e.g., HIV) as well as bacterial or fungal infections, or mayresult from autoimmune disorders. More specifically, infectious diseasescauses by viral, bacterial, fungal or other infection may be treatableusing a protein of the present invention, including infections by HIV,hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malariaspp. and various fungal infections such as candidiasis. Of course, inthis regard, a protein of the present invention may also be useful wherea boost to the immune system generally may be desirable, i.e., in thetreatment of cancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein of the present invention may also to be useful in thetreatment of allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems. Otherconditions, in which immune suppression is desired (including, forexample, organ transplantation), may also be treatable using a proteinof the present invention.

Using the proteins of the invention it may also be possible to immuneresponses, in a number of ways. Down regulation may be in the form ofinhibiting or blocking an immune response already in progress or mayinvolve preventing the induction of an immune response. The functions ofactivated T cells may be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processwhich requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing non-responsiveness or anergyin T cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon reexposure to specific antigen in theabsence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7)), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens.

Upregulation of an antigen function (preferably a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through stimulatingB lymphocyte antigen function may be useful in cases of viral infection.In addition, systemic viral diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration ofstimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express theprotein, and reintroduce the transfected cells into the patient. Theinfected cells would now be capable of delivering a costimulatory signalto, and thereby activate, T cells in vivo.

In another application, up regulation or enhancement of antigen function(preferably B lymphocyte antigen function) may be useful in theinduction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma,lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleicacid encoding at least one peptide of the present invention can beadministered to a subject to overcome tumor-specific tolerance in thesubject. If desired, the tumor cell can be transfected to express acombination of peptides. The transfected tumor cells are returned to thepatient to result in expression of the peptides in the transfected cell.Alternatively, gene therapy techniques can be used to target a tumorcell for transfection in vivo.

The activity of a protein of the invention may, among other means, bemeasured by the following methods: Suitable assays for thymocyte orsplenocyte cytotoxicity include, without limitation, those described in:Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek,D. H. Margulies, E. M. Shevach, W Strober, Pub. Greene PublishingAssociates and Wiley-Interscience (Chapter 3, In Vitro assays for MouseLymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans);Herrmann et al., Proc. Natl. Acad. Sci. U.S.A. 78:2488-2492, 1981;Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500,1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al.,Proc. Natl. Acad. Sci. U.S.A. 78:2488-2492, 1981; Herrmann et al., J.Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572,1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J.Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al.,J. Immunol. 153:3079-3092, 1994. Assays for T-cell-dependentimmunoglobulin responses and isotype switching (which will identify,among others, proteins that modulate T-cell dependent antibody responsesand that affect Th1/Th2 profiles) include, without limitation, thosedescribed in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assaysfor B cell function: In vitro antibody production, Mond, J. J. andBrunswick, M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994. Mixedlymphocyte reaction (MLR) assays (which will identify, among others,proteins that generate predominantly Thl and CTL responses) include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, WStrober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988;Bertagnolli et al., J. Immunol. 149:3778-3783, 1992. Dendriticcell-dependent assays (which will identify, among others, proteinsexpressed by dendritic cells that activate naive T-cells) include,without limitation, those described in: Guery et al., J. Immunol.134:536-544, 1995; Inaba et al., Journal of Experimental Medicine173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad Sci. U.S.A. 88:7548-7551, 1991.

Hematopoiesis Regulating Activity

A protein of the present invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of hematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods: Suitable assays for proliferation anddifferentiation of various hematopoietic lines are cited above. Assaysfor embryonic stem cell differentiation (which will identify, amongothers, proteins that influence embryonic differentiation hematopoiesis)include, without limitation, those described in: Johansson et al.Cellular Biology 15:141-151, 1995; Keller et al., Molecular and CellularBiology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. U.S.A. 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

Tissue Growth Activity

A protein of the present invention also may have utility in compositionsused for bone, cartilage, tendon, ligament and/or nerve tissue growth orregeneration, as well as for wound healing and tissue repair andreplacement, and in the treatment of burns, incisions and ulcers. Aprotein of the present invention, which induces cartilage and/or bonegrowth in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. A protein of this invention may also be used in thetreatment of periodontal disease, and in other tooth repair processes.Such agents may provide an environment to attract bone-forming cells,stimulate growth of bone-forming cells or induce differentiation ofprogenitors of bone-forming cells. A protein of the invention may alsobe useful in the treatment of osteoporosis or osteoarthritis, such asthrough stimulation of bone and/or cartilage repair or by blockinginflammation or processes of tissue destruction (collagenase activity,osteoclast activity, etc.) mediated by inflammatory processes. Anothercategory of tissue regeneration activity that may be attributable to theprotein of the present invention is tendon/ligament formation. A proteinof the present invention, which induces tendon/ligament-like tissue orother tissue formation in circumstances where such tissue is notnormally formed, has application in the healing of tendon or ligamenttears, deformities and other tendon or ligament defects in humans andother animals. Such a preparation employing a tendon/ligament-liketissue inducing protein may have prophylactic use in preventing damageto tendon or ligament tissue, as well as use in the improved fixation oftendon or ligament to bone or other tissues, and in repairing defects totendon or ligament tissue. De novo tendon/ligament-like tissue formationinduced by a composition of the present invention contributes to therepair of congenital, trauma induced, or other tendon or ligamentdefects of other origin, and is also useful in cosmetic plastic surgeryfor attachment or repair of tendons or ligaments. The compositions ofthe present invention may provide an environment to attract tendon- orligament-forming cells, stimulate growth of tendon- or ligament-formingcells, induce differentiation of progenitors of tendon- orligament-forming cells, or induce growth of tendon/ligament cells orprogenitors ex vivo for return in vivo to effect tissue repair. Thecompositions of the invention may also be useful in the treatment oftendinitis, carpal tunnel syndrome and other tendon or ligament defects.The compositions may also include an appropriate sequestering agent as acarrier as is well known in the art.

The protein of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a protein may be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or fasterclosure of non-healing wounds, including without limitation pressureulcers, ulcers associated with vascular insufficiency, surgical andtraumatic wounds, and the like. It is expected that a protein of thepresent invention may also exhibit activity for generation orregeneration of other tissues, such as organs (including, for example,pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,skeletal or cardiac) and vascular (including vascular endothelium)tissue, or for promoting the growth of cells comprising such tissues.Part of the desired effects may be by inhibition or modulation offibrotic scarring to allow normal tissue to regenerate. A protein of theinvention may also exhibit angiogenic activity. A protein of the presentinvention may also be useful for gut protection or regeneration andtreatment of lung or liver fibrosis, reperfusion injury in varioustissues, and conditions resulting from systemic cytokine damage. Aprotein of the present invention may also be useful for promoting orinhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove. The activity of a protein of the invention may, among othermeans, be measured by the following methods: Assays for tissuegeneration activity include, without limitation, those described in:International Patent Publication No. WO95/16035 (bone, cartilage,tendon); International Patent Publication No. WO95/05846 (nerve,neuronal); International Patent Publication No. WO91/07491 (skin,endothelium). Assays for wound healing activity include, withoutlimitation, those described in: Winter, Epidermal Wound Healing pps.71-112 (Maibach, H I and Rovee, D T, eds.), Year Book MedicalPublishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.Invest. Dermatol 71:382-84 (1978).

Inflammation

Proteins of the present invention may also exhibit anti-inflammatoryactivity. The anti-inflammatory activity may be achieved by providing astimulus to cells involved in the inflammatory response, by inhibitingor promoting cell-cell interactions (such as, for example, celladhesion), by inhibiting or promoting chemotaxis of cells involved inthe inflammatory process, inhibiting or promoting cell extravasation, orby stimulating or suppressing production of other factors which moredirectly inhibit or promote an inflammatory response. Proteinsexhibiting such activities can be used to treat inflammatory conditionsincluding chronic or acute conditions), including without limitationinflammation associated with infection (such as septic shock, sepsis orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Proteins of the inventionmay also be useful to treat anaphylaxis and hypersensitivity to anantigenic substance or material.

Tumor Inhibition Activity

In addition to the activities described above for immunologicaltreatment or prevention of tumors, a protein of the invention mayexhibit other anti-tumor activities. A protein may inhibit tumor growthdirectly or indirectly (such as, for example, via ADCC). A protein mayexhibit its tumor inhibitory activity by acting on tumor tissue or tumorprecursor tissue, by inhibiting formation of tissues necessary tosupport tumor growth (such as, for example, by inhibiting angiogenesis),by causing production of other factors, agents or cell types whichinhibit tumor growth, or by suppressing, eliminating or inhibitingfactors, agents or cell types which promote tumor growth.

Other Activities

A protein of the invention may also exhibit one or more of the followingadditional activities or effects: inhibiting the growth, infection orfunction of, or killing, infectious agents, including, withoutlimitation, bacteria, viruses, fungi and other parasites; effecting(suppressing or enhancing) bodily characteristics, including, withoutlimitation, height, weight, hair color, eye color, skin, fat to leanratio or other tissue pigmentation, or organ or body part size or shape(such as, for example, breast augmentation or diminution, change in boneform or shape); effecting biorhythms or caricadic cycles or rhythms;effecting the fertility of male or female subjects; effecting themetabolism, catabolism, anabolism, processing, utilization, storage orelimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, cofactors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoiefic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

Administration and Dosing

A protein of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinantsources) may be used in a pharmaceutical composition when combined witha pharmaceutically acceptable carrier. Such a composition may alsocontain (in addition to protein and a carrier) diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration.

The pharmaceutical composition of the invention may also containcytokines, lymphokines, or other hematopoietic factors such as M-CSF,GM-CSF, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF,Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. Thepharmaceutical composition may further contain other agents which eitherenhance the activity of the protein or compliment its activity or use intreatment. Such additional factors and/or agents may be included in thepharmaceutical composition to produce a synergistic effect with proteinof the invention, or to minimize side effects. Conversely, protein ofthe present invention may be included in formulations of the particularcytokine, lymphokine, other hematopoietic factor, thrombolytic oranti-thrombotic factor, or anti-inflammatory agent to minimize sideeffects of the cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

A protein of the present invention may be active in multimers (e.g.,heterodimers or homodimers) or complexes with itself or other proteins.As a result, pharmaceutical compositions of the invention may comprise aprotein of the invention in such multimeric or complexed form. Thepharmaceutical composition of the invention may be in the form of acomplex of the protein(s) of present invention along with protein orpeptide antigens. The protein and/or peptide antigen will deliver astimulatory signal to both B and T lymphocytes. B lymphocytes willrespond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunolgobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.

The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No.4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all ofwhich are incorporated herein by reference. As used herein, the term“therapeutically effective amount” means the total amount of each activecomponent of the pharmaceutical composition or method that is sufficientto show a meaningful patient benefit, i.e., treatment, healing,prevention or amelioration of the relevant medical condition, or anincrease in rate of treatment, healing, prevention or amelioration ofsuch conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein of the present invention isadministered to a mammal having a condition to be treated. Protein ofthe present invention may be administered in accordance with the methodof the invention either alone or in combination with other therapiessuch as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors. Administration ofprotein of the present invention used in the pharmaceutical compositionor to practice the method of the present invention can be carried out ina variety of conventional ways, such as oral ingestion, inhalation,topical application or cutaneous, subcutaneous, intraperitoneal,parenteral or intravenous injection. Intravenous administration to thepatient is preferred.

When a therapeutically effective amount of protein of the presentinvention is administered orally, protein of the present invention willbe in the form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%protein of the present invention, and preferably from about 25 to 90%protein of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein of the present invention, and preferably from about 1to 50% protein of the present invention.

When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

The amount of protein of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of protein of the present inventionand observe the patient's response. Larger doses of protein of thepresent invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 .mu.g to about 100 mg (preferably about 0.1 ng toabout 10 mg, more preferably about 0.1 .mu.g to about 1 mg) of proteinof the present invention per kg body weight. The duration of intravenoustherapy using the pharmaceutical composition of the present inventionwill vary, depending on the severity of the disease being treated andthe condition and potential idiosyncratic response of each individualpatient. It is contemplated that the duration of each application of theprotein of the present invention will be in the range of 12 to 24 hoursof continuous intravenous administration. Ultimately the attendingphysician will decide on the appropriate duration of intravenous therapyusing the pharmaceutical composition of the present invention.

Protein of the invention may also be used to immunize animals to obtainpolyclonal and monoclonal antibodies which specifically react with theprotein. Such antibodies may be obtained using either the entire proteinor fragments thereof as an immunogen. The peptide immunogensadditionally may contain a cysteine residue at the carboxyl terminus,and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).Methods for synthesizing such peptides are known in the art, forexample, as in R. P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963);J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987). Monoclonalantibodies binding to the protein of the invention may be usefuldiagnostic agents for the immunodetection of the protein. Neutralizingmonoclonal antibodies binding to the protein may also be usefultherapeutics for both conditions associated with the protein and also inthe treatment of some forms of cancer where abnormal expression of theprotein is involved. In the case of cancerous cells or leukemic cells,neutralizing monoclonal antibodies against the protein may be useful indetecting and preventing the metastatic spread of the cancerous cells,which may be mediated by the protein.

For compositions of the present invention which are useful for bone,cartilage, tendon or ligament regeneration, the therapeutic methodincludes administering the composition topically, systematically, orlocally as an implant or device. When administered, the therapeuticcomposition for use in this invention is, of course, in a pyrogen-free,physiologically acceptable form. Further, the composition may desirablybe encapsulated or injected in a viscous form for delivery to the siteof bone, cartilage or tissue damage. Topical administration may besuitable for wound healing and tissue repair. Therapeutically usefulagents other than a protein of the invention which may also optionallybe included in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. Preferably for bone and/orcartilage formation, the composition would include a matrix capable ofdelivering the protein-containing composition to the site of bone and/orcartilage damage, providing a structure for the developing bone andcartilage and optimally capable of being resorbed into the body. Suchmatrices may be formed of materials presently in use for other implantedmedical applications. The choice of matrix material is based onbiocompatibility, biodegradability, mechanical properties, cosmeticappearance and interface properties. The particular application of thecompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sintered hydroxapatite,bioglass, aluminates, or other ceramics. Matrices may be comprised ofcombinations of any of the above mentioned types of material, such aspolylactic acid and hydroxyapatite or collagen and tricalciumphosphate.The bioceramics may be altered in composition, such as incalcium-aluminate-phosphate and processing to alter pore size, particlesize, particle shape, and biodegradability. Presently preferred is a50:50 (mole weight) copolymer of lactic acid and glycolic acid in theform of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix. A preferred family of sequestering agents is cellulosicmaterials such as alkylcelluloses (including hydroxyalkylcelluloses),including methylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins ofthe invention may be combined with other agents beneficial to thetreatment of the bone and/or cartilage defect, wound, or tissue inquestion. These agents include various growth factors such as epidermalgrowth factor (EGF), platelet derived growth factor (PDGF), transforminggrowth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growthfactor (IGF). The therapeutic compositions are also presently valuablefor veterinary applications. Particularly domestic animals andthoroughbred horses, in addition to humans, are desired patients forsuch treatment with proteins of the present invention. The dosageregimen of a protein-containing pharmaceutical composition to be used intissue regeneration will be determined by the attending physicianconsidering various factors which modify the action of the proteins,e.g., amount of tissue weight desired to be formed, the site of damage,the condition of the damaged tissue, the size of a wound, type ofdamaged tissue (e.g., bone), the patient's age, sex, and diet, theseverity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA). Cells may alsobe cultured ex vivo in the presence of proteins of the present inventionin order to proliferate or to produce a desired effect on or activity insuch cells. Treated cells can then be introduced in vivo for therapeuticpurposes.

The following examples are provided to further illustrate particularembodiments of the invention, and are not to be construed as limitingthe scope of the present invention.

EXAMPLE 1 Isolation of Polynucleotides Encoding Novel Kinases

High through put sequencing of murine dendritic cell, murine lymph nodestromal cell, and human dendritic cell libraries generated nucleotidesequences which were used to query public and private sequence databasesusing an alogithim designed to recognize kinase subdomains. Putativekinase clones identified in this manner were further sequenced. Theresultant complete clone sequences are shown as: SEQ ID NO:1-6, andTable VIII identifies the specific library and clones for each of thesesequences. In the case of SS4694 (SEQ ID NO:6), sequence informationderived from SS4694 and KIAA0551 (see pp. ////26-27) enabled the cloningof LNRK-1 (SEQ ID NO:7) from a Marathon-ready human spleen cDNA library(Clontech).

TABLE VIII Name SEQ ID NO Library Clone # MDCK-1 1 Murine dendritic990219MDCA001250HT cell MDCK-2 2 Murine dendritic 990205MDCA999084HTcell MDCK-3 3 Murine dendritic 990217MDCA001428HT cell MLSK-1 4 Murinelymph 980906MLSA002022HT node stromal cell MLSK-2 5 Murine lymph9980905MLSA001070HT node stromal cell LNRK-1 6 Human dendritic ss4694cell

EXAMPLE 2 Use of Kinase Polypeptides in an ELISA

Kinase-specific ELISA:

Serial dilutions of kinase-containing samples (in 50 mM NaHCO₃, broughtto pH 9 with NaOH) are coated onto Linbro/Titertek 96 well flat bottomE.I.A. microtitration plates (ICN Biomedicals Inc., Aurora, Ohio) at100:1/well. After incubation at 4° C. for 16 hours, the wells are washedsix times with 200:1 PBS containing 0.05% Tween-20 (PBS-Tween). Thewells are then incubated with FLAG®-binding partner at 1 mg/ml inPBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100:1 perwell), followed by washing as above. Next, each well is incubated withthe anti-FLAG® (monoclonal antibody M2 at 1 mg/ml in PBS-Tweencontaining 5% FCS for 90 minutes (100:1 per well), followed by washingas above. Subsequently, wells are incubated with a polyclonal goatanti-mIgGI-specific horseradish peroxidase-conjugated antibody (a 1:5000dilution of the commercial stock in PBS-Tween containing 5% FCS) for 90minutes (100:1 per well). The HRP-conjugated antibody is obtained fromSouthern Biotechnology Associates, Inc., Birmingham, Ala. Wells then arewashed six times, as above.

For development of the ELISA, a substrate mix [100:1 per well of a 1:1premix of the TMB Peroxidase Substrate and Peroxidase Solution B(Kirkegaard Perry Laboratories, Gaithersburg, Md.)] is added to thewells. After sufficient color reaction, the enzymatic reaction isterminated by addition of 2 N H₂SO₄ (50:1 per well). Color intensity(indicating kinase/binding partner binding activity) is determined bymeasuring extinction at 450 nm on a V Max plate reader (MolecularDevices, Sunnyvale, Calif.).

EXAMPLE 3 Monoclonal Antibodies That Bind

This example illustrates a method for preparing monoclonal antibodiesthat bind kinases of the invention. Suitable immunogens that may beemployed in generating such antibodies include, but are not limited to,purified kinase polypeptides or an immunogenic fragment thereof, orfusion proteins containing a kinase of the invention (e.g., a solublekinase/Fc fusion protein).

Purified kinases can be used to generate monoclonal antibodiesimmunoreactive therewith, using conventional techniques such as thosedescribed in U.S. Pat. No. 4,411,993. Briefly, mice are immunized withkinase immunogen emulsified in complete Freund's adjuvant, and injectedin amounts ranging from 10-100 μg subcutaneously or intraperitoneally.Ten to twelve days later, the immunized animals are boosted withadditional kinase immunogen emulsified in incomplete Freund's adjuvant.Mice are periodically boosted thereafter on a weekly to bi-weeklyimmunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision to test for anti-kinaseantibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay)or inhibition of kinase/binding partner binding.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of kinase immunogen insaline. Three to four days later, the animals are sacrificed, spleencells harvested, and spleen cells are fused to a murine myeloma cellline, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusionsgenerate hybridoma cells, which are plated in multiple microtiter platesin a HAT (hypoxanthine, aminopterin and thymidine) selective medium toinhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified kinase polypeptides by adaptations of the techniques disclosedin Engvall et al., Immunochem. 8:871, 1971 and in U.S. Pat. No.4,703,004. A preferred screening technique is the antibody capturetechnique described in Beckmann et al., (J. Immunol. 144:4212, 1990)Positive hybridoma cells can be injected intraperitoneally intosyngeneic BALB/c mice to produce ascites containing high concentrationsof anti-kinase monoclonal antibodies. Alternatively, hybridoma cells canbe grown in vitro in flasks or roller bottles by various techniques.Monoclonal antibodies produced in mouse ascites can be purified byammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to Protein A or Protein G can also be used, as canaffinity chromatography based upon binding to kinase polypeptides of theinvention.

EXAMPLE 4 PCR Analysis of Tissue-Specific Kinase Expresion

The tissue distribution of MDCK-2, MDCK-3, MLSK-1, MLSK-2 and ss4694mRNA was investigated. Using Clonetech multiple tissue cDNA panels andclone specific oligonucleotide PCR primers, we determined thatexpression of MDCK-2 was ubiquitous throughout the Clontech murine cDNApanel, with highest levels in spleen, liver, skeletal muscle and sevenday embryo. MDCK-3 expression was highest in heart, brain and spleen;lower level expression was seen in liver, skeletal muscle and testis,and low but detectable levels in lung and kidney. Expression of MLSK-1was found to be highest in the heart, lung, and liver.

Lower levels of MLSK-1 were detected in the kidney and skeletal muscle,and MLSK-1 expression was absent from brain and spleen. Expression ofMLSK-2 was highest in the heart and spleen while lower levels ofexpression were found in the lung and liver, and no expression wasdetectable in the brain, skeletal muscle, kidney and testis.

SS4694 and therefore LNRK-1 expression is ubiquitous throughout thehuman cDNA panels (Clontech human tissue panel 1, human tissue panel 2and human immune tissue panel) with highest levels in peripheral bloodlymphocyte cDNA. All other tissues had roughly equivalent levels ofexpression.

EXAMPLE 5 Measuring Kinase Activity

Isolated kinase polypeptides or fusion proteins containing the isolatedprotein kinase domain can be used in an assay of protein kinaseactivity.

Typically this would be carried out by combining a kinase of theinvention with radiolabeled ATP (γ³²P-ATP) and a magnesium (or otherdivalent cation, such as manganese) salt in buffer solution containing apeptide or protein substrate. Peptide substrates are generally from 8-30amino acids in length and may terminate at the N- or C-terminus withthree or more lysine or arginine residues to facilitate binding of thepeptide to phosphocellulose paper. The substrate may also be a proteinknown to be phosphorylated readily by a kinase of the invention. Manysuch general kinase substrates are known, such as, α or β casein,histone H1, myelin basic protein, etc. After incubation of this reactionmixture at 20-37° C. for a suitable time, the transfer of radioactivephosphate from ATP to the substrate protein or substrate peptide may bemonitored, by acidifying the reaction mixture then spotting it ontophosphocellulose paper, and subsequent washing of the paper with adilute solution of phosphoric acid, in the case of a peptide substrate,or by application of the reaction products to a gel electrophoresissystem followed by autoradiographic detection in the case of proteins.

A specific example of this type of assay of kinase activiy is thePhosphoSpots™ assay (Jerini Bio Tools GMBH), in which protein or peptidesubstrates are attached to a solid support. In one example, thesubstrates may be peptides where each is known to be phosphorylated by aparticular kinase. When the kinase being tested is added to thesubstrates in the presence of γ-³²P, any attached proteins or peptidesthat are suitable subtrates for that kinase will be labeled with theγ-³²P which can be quantitatively detected using a phosphoimager. (See,for example, Tegge et al., 1995, Determination of cyclicnucleotide-dependent protein kinase substrate specificity by the use ofpeptide libraries on cellulose paper, Biochemistry 34 (33): 10569-10577,which is incorporated by reference herein). The results of such an assayfor MLSK-1 substrate specificity are shown in Table IX below.

TABLE IX Phosphorylation of known kinase substrates by MLSK-1 Phosphory-SEQ lation Known PK Substrate ID by MLSK-1 Protein Kinase (“PK”)Sequence NO: (% of max.) cAMP-dependent LRRASLG 17 65.9 protein kinasecGMP-dependent RKISASEFDRPLR 18 62.2 protein kinase protein kinase CKKRFSFKKSFKLSGFSFK 19 100.0 protein kinase C QKRPSQRSK 20 75.0Ca-/calmodulin- KKALRRQETVDAL 21 20.9 dependent PK Ca-/calmodulin-PLARTLSVAGLPGK 22 10.3 dependent PK casein kinase II RRRDDDSDDD 23 17.6cdc2-kinase Ac-SPGRRRRK 24 94.8 p34 cdc-kinase PKTPKKAKKL 25 82.0p42/p44 MAP APRTPGGRR 26 88.6 kinase p42/p44 MAP EAAEAEPAEPSSPAAEAEGA 270.0 kinase p42/p44 MAP LMECRNSPVAKT 28 3.8 kinase casein kinase IRRKDLHDDEEDEAMSITA 29 14.1 S6 kinase LSSLRASTSKSGGQK 30 66.6 myosinlight KKRPQRATSNVFS 31 72.0 chain kinase insulin receptorKKKLPATGDYMNMSPVGD 32 33.7 tyrosine kinase csk tyrosine KKKKEEIYFFF 3382.1 kinase raf-1 kinase SGQLIDSMANSFVGTRS 34 43.1 abl tyrosineEAIYAAPFAKKK 35 69.3 kinase c-src tyrosine YIYGSFK 36 66.6 kinase

Kinase activity may also be measured, in vitro or in intact cells, usinga fluorescence resonance energy transfer (FRET) assay, in which thetransfer of energy between fluorescently tagged kinase and substratemolecules is detected. (For example, see Ng et al., 1999, Imagingprotein kinase C alpha activation in cells, Science 283 (5410):2085-2089, which is incorporated by reference herein.)

Other methods are available to conveniently measure the kinase-mediatedtransfer of phosphate to substrate proteins or peptides, such as thescintillation proximity assay, and the use of monoclonal antibodies thatare specific for phosphorylated or non-phosphorylated forms of substratemolecules; these methods are well known to those practiced in the art.

EXAMPLE 6 Tissue- and Stge-Specific Expression of Kinase mRNAs

The expression of MDCK-2, MDCK-3, MLSK-1, MLSK-2, and ss4694 (LNRK-1)was assessed by PCR using gene-specific oligonucleotide primers directedtoward these kinase-encoding polynucleotides usign Clontech multipletissue cDNA panels as templates. Expression of MDCK-2, MDCK-3, MLSK-1,and MLSK-2 was assayed using the murine cDNA panel (see Table X below).Expression of MDCK-2 was observed in all tissues on this panel, with thehighest levels in spleen, liver, skeletal muscle, and seven-day embryo.Expression of MDCK-3 was highest in heart, brain and spleen; lower-levelexpression was seen in liver, skeletal muscle and testis, and low butdetectable levels were observed in lung and kidney; however, expressionof MDCK-3 was completely absent from murine embryo. Expression of MLSK-1was highest in the heart, lung, and liver, and is absent from the brainand spleen; MLSK-1 expression in the embryo is only seen at the 11-daystage. Expression of MLSK-2 is found to be highest in the heart andspleen. MLSK-2 is expressed at lower levels in lung, liver, and 7-dayembryo, and is not detectable in brain, skeletal muscle, kidney, testis,or embryonic tissue at other stages of development. ss4694(LNRK-1)expression is ubiquitous throughout the human cDNA panels, with highestlevels in PBL (peripheral blood lymphocyte) cDNA. All other tissues hadroughly equivalent levels of expression.

TABLE X MURINE PANEL MDCK-2 MDCK-3 MLSK-1 MLSK-2 Heart ** *** *** ***Brain ** *** Spleen *** *** ** Lung ** * *** * Liver *** ** *** *Skeletal Muscle *** ** * Kidney ** * ** Testis ** ** 7-day Embryo *** *11-day Embryo ** ** 15-day Embryo * 17-day Embryo **

Expression of the ss4694 clone (and therefore of LNRK-1) was assayedusing the Clontech “human tissue” and “human immune” cDNA panels (seeTable XI below). ss4694 (LNRK-1) expression is observed throughout thehuman cDNA panels, with highest levels in PBL (peripheral bloodlymphocyte) cDNA. All other tissues had roughly equivalent levels ofexpression.

TABLE XI ss4694/LNRK-1 Expression HUMAN TISSUE PANEL I Brain ** Heart **Kidney * Liver ** Lung ** Pancreas ** Placenta ** Skeletal Muscle *HUMAN TISSUE PANEL II Colon ** Ovary ** PBL *** Prostate ** SmallIntestine * Spleen ** Testis ** Thymus ** HUMAN IMMUNE PANEL Bone Marrow** Fetal Liver ** Lymph Node ** PBL *** Spleen ** Thymus ** Tonsil **

EXAMPLE 7 Regulation of MDCK-3 Expression in Dendritic Cell Maturation

The regulation of MDCK-3 RNA expression during dendritic cell“maturation” and/or “activation” was assayed by RT (reversetranscriptase) PCR (polymerase chain reaction). Flt3 ligand (“Flt3L”) isa growth factor that stimulates the proliferation of hematopoieticcells. RNA samples isolated from splenic dendritic cells purified fromFlt3L-treated mice were assayed by RT PCR using primers derived from theMDCK-3 cDNA sequence. MDCK-3 RNA was undetected in the freshly isolateddendritic cells, but was present in cells following overnight culture ina defined medium, a growth procedure which causes dendritic cell“maturation”. This indicates that regulation of MDCK-3 expression iscorrelated with the maturation state of at least one type of cellinvolved in immune responses.

Furthermore, RT PCR was performed on RNA isolated from a differentdendritic cell system, where murine bone marrow cells are isolated andthen cultured in media containing Flt3 ligand for nine days, with theaddition of various dendritic cell “activators” during the final 24hours of the incubation period. MDCK-3 mRNA levels were specificallyaltered by some “activators” but not others.

These assays can also be performed with a variety of cells such asperipheral blood mononuclear cells and a variety of activatingsubstances such as IL-4, GM-CSF, TNF, IL-2, IFN, LPS, etc. to test for acorrelation between changes in cell differentiation or proloferativeactivity and the expression of the kinases of the invention.

EXAMPLE 8 Assaying Activation of Kinase Pathways by DetectingPhosphorylation of Known Pathway Components

Cell signalling pathways often involve a cascade of phosphorylationevents. Over-expression of kinases in cells can activate such signallingpathways, and this activation may be detected by measuring the level ofphosphorylation of molecules that are known to be ‘downstream’phosphorylated recipients of ‘upstream’ kinase activity. Conversely,over-expression of a catalytically inactive, truncated, or otherwisemutated form of a kinase can act as a dominant negative mutation anddisrupt or abolish normal signalling events ‘downstream’ of the kinase.

In one example, we expressed active forms of MLSK-1 and of MLSK-2 andshowed that when over-expressed in COS cells each of these kinasesactivates the MAP kinase signaling pathway as evidenced by thegeneration of phosphorylated forms of ERK, a ‘downstream’ kinase in theMAP kinase pathway. Phosphorylation of signalling pathway molecules canbe detected in a variety of ways, including incorporation of ³²Pfollowed by immunoprecipitation, FRET assays as described in Example 5,or the use of phosphorylation-state-specific antibodies in ELISA assaysor on Western blots. Additionally, kinase specificity for a particularcell signalling pathway can be assessed by comparing the phosphorylationresponses of ‘downstream’ molecules in different pathways toover-expression of that kinase. For example, over-expression of MLSK-1and of MLSK-2 in COS cells had no effect on the stress-activated kinasepathway, as expression of these kinases did not result in activation ofeither JNK or p38 kinases.

EXAMPLE 9 Reporter Gene Assavs of Kinase Pathway Activation

Activation of cell signalling pathways by kinases of the invention mayalso be assayed using reporter gene constructs. In such constructs areporter gene such as luciferase or β-galactosidase is placed downstreamof a promoter, enhancer, or other transcriptional regulatory elementthat is known to bind transcription factors as a result of activation ofa cell signalling pathway. This transcriptional regulatory element maybe selected on the basis of its known association with a relevanttranscription factor, such as AP-1 or NFκB, or on the basis of physicalassociation with a downstream gene known to be regulated by thesignalling pathway. An example of the use of such reporter constructs isdescribed in Ling et al., 1998, NF-kappaB-inducing kinase activatesIKK-alpha by phosphorylation of Ser-176, Proc Natl Acad Sci USA 95 (7):3792-3797, which is incorporated by reference herein.

In one example, assays to determine whether MLSK-1 or MLSK-2 couldactivate the transcription factor AP-1 were performed. AP-1 is atranscription factor known to be involved in the JNK and p38 signallingpathways. MLSK-1 and MLSK-2 were each individually co-tranfected with anAP-1-luciferase construct into COS-7 cells in a standard AP-1-luciferasereporter assay. The overexpression of neither MLSK-1 nor MLSK-2activates AP-1 using this assay system, consistent with these kinasesnot participating in the stress-activated JNK and p38 signallingpathways.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments of the inventionand should not be construed to limit the scope of the invention. Theskilled artisan readily recognizes that many other embodiments areencompassed by the invention.

36 1 896 DNA Mus musculus 1 ttggaacgag acgacctgct cggcggcagc ggagtgagcgagccggagcg tgagtggccc 60 cgcggcggcc atgggcgacc cagcccccgc ccgcagcctggacgacatcg acctgtctgc 120 cctgcgggac cctgcaggaa tctttgagct ggtggaggtggttggcaatg gaacctatgg 180 acaggtatac aaggggcggc acgtcaagac tgggcagctggctgccatta aggtcatgga 240 tgtcacagag gatgaggagg aagagatcaa acaggaaatcaacatgttaa agaagtactc 300 tcaccatcgc aatattgcca cctactatgg ggcctttatcaagaagagcc ctcctgggaa 360 cgatgaccag ctctggctgg tgatggagtt ctgcggtgctggttcagtga ccgacctggt 420 aaagaacaca aaagggaacg cactgaagga ggattgcattgcttacatct gcagggagat 480 tctcaggggt cttgcccatc tccatgccca caaggtgatccacagagata tcaagggaca 540 aaatgtgctg ctgacagaga atgctgaagt caagctagtggattttgggg tgagtgctca 600 gctggaccgc actgtgggca ggcggaacac tttcattggaaccccatact ggatggctcc 660 agaagtcatt gcctgtgacg agaaccccga tgccacctatgactacagga gtgacatttg 720 gtctctagga atcacagcca ttgaaatggc agagggagccccccctctgt gtgacatgca 780 ccctatgcgg gccctcttcc tcatccctcg gaaccctccccccaggctca agtcaaagaa 840 atggtctaag aagttcactg acttcatcga cacgtgtctcatcaagactt acctga 896 2 1060 DNA Mus musculus 2 cggcgcctgc agtggcgcggggagaggcga tcggccccgg atcccgcagc cctgcagccc 60 gcgtagaccg agctgccccggcgccccgaa tcctgaagtc ccagttgagt cagaatggat 120 gaacaatcac aaggaatgcaagggccgccc gttactcagt tccagccaca gaaggcatta 180 cggccagata tgggctataatactttagcc aacttccgaa tagaaaagaa aattggtcgt 240 ggacaattta gtgaagtttatagagcatcc tgtctcttgg atggagtgcc ggtagcgtta 300 aaaaaagtac agatatttgatttaatggat gccaaagcac gtgctgattg tatcaaagaa 360 atagacctcc ttaagcaactcaaccatcca aatgtaatta aatactatgc atcattcatt 420 gaggataatg agctgaacatagttttggag ttagcagatg ctggtgacct ctccagaatg 480 ataaagcact ttaagaaacaaaagaggcta atccctgaga gaaccgtttg gaaatacttc 540 gttcagctct gcagtgcactggaccacatg cattctcgaa gagtcatgca cagagatata 600 aaaccagcta atgtgttcattacagccact ggggtagtaa aactcggaga ccttgggctt 660 ggtcggtttt tcagctccaaaaccacagct gcacattctt tagtgggtac accttactac 720 atgtctccag agagaatacatgaaaatgga tacaacttca agtctgacat ctggtctctt 780 ggctgtctgc tatatgagatggctgcactg cagagtcctt tctacggcga caagatgaac 840 ttgtattctc tgtgtaagaagatagagcag tgtgactacc cgcctctccc gtcagatcac 900 tattcggagg agctacgacagctagttaat atatgcatca acccagatcc agagaagcga 960 cccgacatcg cctatgtttatgatgtggca aagaggatgc atgcatgtac cgcaagcacc 1020 taaactgtac aagatcctgaagcaggtcgt ctcgttccaa 1060 3 1694 DNA Mus musculus 3 tggaacgagacgacctgctc cggagagcag tagaaattag agccagggag ggaccgcggc 60 ggcagcagccaaggcgaaag aggaaactgc cgaagaggaa gctctgcggc agcccgagcc 120 tccctgcgctccgcatcctc ccgctccgca tcccggcgcc gggcatcccc cggagcccgc 180 gccgcgcctccggcgcccct tccccagcgc aaccctcggc cgccctacag cattagtctg 240 ccatggcccgggagaacggc gagagcagct cctcctggaa aaagcaagca gaagacatta 300 agaagatcttcgagttcaag gagaccctcg gaactggggc cttttctgaa gttgttttag 360 ccgaggagaaagctactggg aagctcttcg cagtgaagtg catcccgaag aaggcgctga 420 agggcaaggagagcagcatc gagaacgaga ttgccgtgct tagaaagatt aagcatgaaa 480 acattgttgccttggaagat atttatgaaa gcccaaatca cctctacctg gtcatgcaac 540 ttgtgtctggtggagaactc ttcgatcgga tagtggagaa ggggttttac acagagaaag 600 atgccagcactctcatccgc caggtcctgg atgccgtata ctatctccac agaatgggca 660 ttgtccacagggacctcaag ccggagaatc tcttatacta cagtcaagac gaggagtcca 720 aaataatgatcagtgacttt ggcttgtcga aaatggaggg caaaggagat gtgatgtcca 780 cggcctgcgggaccccaggc tatgttgctc cggaagttct cgcccagaaa ccgtacagca 840 aagctgtggactgctggtcc atcggggtga tcgcctatat cttgctctgt ggttaccctc 900 ctttttatgatgaaaatgac tcgaagctgt ttgaacagat cctcaaggca gaatatgagt 960 ttgattccccctactgggat gacatctccg actctgccaa agacttcatt cggaatctga 1020 tggagaaagacccaaataaa agatacactt gtgagcaggc agctcgacac ccatggattg 1080 ctggtgacacagcccttagc aaaaacattc acgaatctgt cagtgcccag atccggaaga 1140 attttgcaaagagcaaatgg agacaagcgt ttaacgccac ggcagtcgtg agacatatgc 1200 ggaggctccagcttggcagc agcctggaca gttcaaatgc aagtgtctca agcaacctca 1260 gtttggccagccaaaaagat tgtgcgtctg gcaccttcca cgctctgtag tttcctttct 1320 tcttcgtcgggggtcgcagg attcggagct gagaggagac ccaggcccac cactgtgaca 1380 acagggcacactggaagcaa gtgacccggc tctggaggtg gaacccaggg ggcagggccg 1440 gggaaggagaagcccctggc cggagcagct cctgcatcag aaaccccacc caccctgcat 1500 ggtgcacctgcataggactg gaagatagaa ggttttttat ggccatattt tatactgcaa 1560 ttctgatgtgttcatttctc acaaactgta ctgactgact caaggggagc tggcgtcacg 1620 ggatctggtgctgtatataa gaatcttgca aagctctaac tgaatggacc ttctagcagg 1680 tcgtctcgttccaa 1694 4 2902 DNA Mus musculus 4 cactagtgga tccaaagaat tcggcacgaggcgtgctcgg gtgcggctgt gacctctgag 60 cccgcggctc agcgcgcgct gctactgctgcccgacccac tccacctcgc ggtccccgca 120 ccatggagtc ggtggcctta ctccagcgcccgagccaggc tccctcggcc tccgccctgg 180 cctcggagag cgcccggccg ctggcggacgggctcatcaa gtcgcctaaa cctctgatga 240 agaagcaggc ggtgaagcgg caccatcacaaacacaacct gcggcaccgc tacgagttcc 300 tggagacgct gggcaagggc acctacgggaaggtgaagaa ggcacgagag agctcggggc 360 gtctggtggc catcaagtcc atcaggaaagacaaaatcaa agatgagcag gatctgctgc 420 acatacggag ggagattgag atcatgtcttcactcaacca cccccacatc attgccatcc 480 atgaagtgtt tgagaatagc agcaagattgtgattgtcat ggagtatgcc agccgaggcg 540 atctgtatga ttacatcagt gagcggccacggctgagtga gcgggacgcc aggcatttct 600 tccgacagat cgtgtctgcc ctgcactactgccaccagaa cgggatcgtt caccgagatc 660 tcaagctgga aaacatcctt ctagatgccaatggaaacat caagattgct gactttggcc 720 tctccaacct gtaccacaaa ggcaagttcctccagacgtt ctgtgggagc cctctctacg 780 cctcgcctga gatagtcaac gggaagccctatgtgggccc agaggtggac agctggtctc 840 tgggcgttct cctgtacatc ctggtgcatggcaccatgcc ctttgacggg caggatcata 900 aaacactggt gaagcaaatc agtaacggggcttaccgtga gccgcccaag ccgtccgatg 960 cctgtggcct gatccggtgg ctgttaatggtgaaccccac ccgtcgggcc acactggagg 1020 atgtagccag tcattggtgg gtcaactggggttacaccac cggagtcggg gaacaggaag 1080 ccctgcgtga gggtgggcac cctagtggtgactttggccg ggcctccatg gcggactggt 1140 tacgtcgctc ctcgcgcccc ctcctggagaatggagccaa ggtgtgcagc ttcttcaagc 1200 agcacgtgcc gggaggtgga agcactgtacctgggctgga gcggcaacat tctcttaaga 1260 agtcccgaaa ggagaatgac atggctcaaaatctgcaagg tgacccggct gaggatacct 1320 cttctcgccc tggcaagagc agccttaagcttccgaaagg cattctcaag aaaaagtcct 1380 ctacctcgtc aggggaggta caggaggaccctcaggaact cagaccggtg cctgatactc 1440 cagggcagcc tgtccctgct gtatccctgctcccaaggaa aggcatcctt aagaagtctc 1500 gacagcgtga atctggttac tactcctctccagagcccag cgagtctggg gaactcttag 1560 acgccagtga tgtgtttgtg agtggggaccccgtggagca gaagtctcca caggcttcag 1620 ggctcctcct ccaccgcaag ggcattctcaaactcaatgg caagttctcc cgcacagcct 1680 tagaaggcac tacccctagc acctttggctccctggacca actggcctcc tcccatcctg 1740 cagcccggcc cagccgcccc tcaggggctgtgagtgagga cagcatcctg tcctccgagt 1800 cctttgacca attggacttg cctgaacgtcttcccgaaac cccactgagg ggctgtgtgt 1860 ctgtggacaa cctgaggggg cttgagcagcctccctcaga aggtctgaag cgatggtggc 1920 aggaatcctt gggggatagc tgcttttctctgacagactg ccaagaggtg actgcagcct 1980 acagacaagc cctaggaatc tgctcaaagctcagctgagg aagggagatg gtgccctagt 2040 atggggtagg ctctgagagg gtttgcagaggaaccctggg tcggattcct ccagtgaata 2100 gagtacatca agggctctac gtctgcagcctgactgaacc tgaaagatga gagaaatcgc 2160 attgatgtgg aaaggaatgg gaacccttgctgcccgagtg ttatagtggg gtggcctgaa 2220 ggtgcctacc tcctttgtgc catgagtgtcacccatgaca tttcccaccc tgttctctgg 2280 ctgcaccttc acataagttt ctgtttccatcaaccaccag ggttagaacc ctgacttcct 2340 gggaggtaat gtgtagtgac tgccattatttagagaggaa acagcctctg gtttccatct 2400 ctgctgctgt gcatctcaaa gacctgggaagactcggacc gctgtttgac ttcatctcaa 2460 ggggaccaga tgcccctgga ccccatcttagatctcagag acttgaacct tgaagctgtt 2520 cctagtaccc agatgtggat ggatgctctgtttctcaggc caacgggacc tagaatgtgc 2580 tgacttattt atttttttgt gattctcacttctgtttttt ggtttttgtt tgtttgtttg 2640 tttttgtttt taagtgaatt ttgctgctttcaataatgtg aatgctgtgt tctggggaac 2700 tccactgtgc cactgaagtt tatgtacagagaagtatttg gcaatgatgt ccctctattc 2760 aaggggggtg ggggcgtttt tcaaatgtatgtcttgagca ctgtctggat tgagtctcca 2820 gtcccttcac acccaaggct ggccaccctccctcatcttc atctgtggcc aaaaaaaaaa 2880 aaaaaaaaaa aaaaaaaaaa aa 2902 53228 DNA Mus musculus 5 tggatccaaa gaattcggca cgaggcggag tcccgcctcgccgcccctcg agcgccccca 60 gcttctctgc tggccggaac ctgcaccccg aaccaggaagcacctggcgg cgggcgcggg 120 atggctgggc ccagctgggg tctccctcgg ctggacggtttcatccttac cgagcgcctg 180 ggcagtggca cgtacgccac ggtgtacaag gcctacgccaagaaggatac tcgggaagtg 240 gtagccataa aatgcgtggc caagaagagt ctcaacaaggcgtcagtgga aaacctcctg 300 actgagattg agatcctcaa gggcattcgg cacccccatatcgtgcagct gaaagacttc 360 cagtgggaca atgacaatat ctacctcatc atggagttctgtgcaggggg tgacctgtct 420 cgcttcattc atacccgcag gatcctgcct gagaaggtggcccgtgtttt catgcagcag 480 ttggctagtg ccctgcagtt cctgcatgaa cgaaacatctctcacttgga tctgaaaccg 540 cagaacatcc tgctgagctc tttggagaag ccccacctgaaactggcaga ctttggcttt 600 gcccagcaca tgtccccgtg ggacgaaaaa cacgtgctccgtggctcccc gctctatatg 660 gctcctgaga tggtgtgtcg gcggcagtat gatgcgcgtgtggacctctg gtctgtgggg 720 gtgatcctgt acgaagccct ctttgggcag cccccctttgcctccagatc gttctcagag 780 ctagaagaaa agattcgcag caatcgggtg attgaggtgcgtctggcagg gtctaggcat 840 ccaccgggga ttgagggact caaggcccag aagtttgttcagcactgcag tgcaggctct 900 gggcgtttca tggcagtggg gcatgttctg tggtggaagcctagagtctg gtccgttcct 960 gaggatccat atcagccacg acaggcaaca aatgaccaggcccaatcttc ccatagtccg 1020 gggctggagg caaataccca tttgatagga gactgataaaggatgcttgg ctctcttcct 1080 gcacatcacc gggacttgcc atgatccact cagattacccacagcaaaca cgtaccctta 1140 tgggggttcc taacaggcct tgggctttgg gctcagatgttggagccttc tgtgatgtgt 1200 ctctgctcta tgcctctgta gctccctctt cggccccaactctccctaga ctgccgggac 1260 ctgttgcagc gacttctaga gcgggacccc gcccgtcgaatctccttcaa ggacttcttt 1320 gcccatccct gggtggacct ggagcacatg cccagtggggagagcctggc acaggcaagg 1380 gcccttgtgg tggaggctgt gaagaaggac caggagggggatgctgccgc tgccctgtcg 1440 ctctactgca aggctctgga cttctttgta cctgcgctacactgtgagaa ccaggccatt 1500 cctataacct gtgtgcagag gggggcagga gttgggtcaggctccccatt cagagcttag 1560 gggagatggt gcagaagatc aacgtggaac tgagtatctgaagattgcaa agggcttact 1620 gtggggtagg ctttcaggac agcatcctca tatgaacccttcaccttctg cagacgaagt 1680 ggatgcccag aggaaggagg caattaaggc gaaggtgggacagtatgtgt cccgggcaga 1740 ggagctcaaa gccattgtct cctcctccaa tcaggccctgctaagacagg gcacaactgt 1800 ccaagagctg cttcgaggct gctccctcac catgagcctttactctcaca tcagagatgg 1860 cccgtgacaa accacgcctc ctggctgccc tggaagtggcctcagctgcc ctggccaagg 1920 aggaggaagc tggcaaagag caggatgccc tggacctgtaccagcacagc ctcggggagc 1980 tgctagtgct gttggcagca gaggccccag gccgaaggcgggagctcctt cacaccgagg 2040 ttcagaacct catggctcga gctgaatacc tgaaggagcagatcaagata agggagtctc 2100 actgggaagc ggagagtctg gacaaagagg ggctgtcggagtctgttcgt agttcttgca 2160 cactgcagtg acaccggaag gagcagcgga tggagcacaaccctagagag aagctgcatt 2220 accaactcag gttgacacct gcacacctgg gaccttcctggacgagcagc tcccacatgc 2280 tggttcccag cattcctctg agtgttctcc acccttggggcgtctggtgg caggtgtact 2340 aagctctggg agaattactt gaatgtgacc ttgtcattaggtgactgctg gtctaagcct 2400 gtccggcttc aggacaccat caccccgttg tgttttgttctgcaaagagg acgtcatgcc 2460 tcttcaggac acttgctacc agacagctgc tgtacctgggccacccctcc ctgggagcct 2520 ttattccaac cctacttttt ttcttgcact ggaatgggacactcggatac cctcagggac 2580 tacctacctg acagtatgct ctcggctctc agacctctccagtcttcctg cgagctcaga 2640 gctgccatcc ttttcagttc tttaagacaa tccttcatgcatgaaagtca tgccctttgt 2700 aaaggtggaa tacatgtgag aaccccagac cttccctgccttggcatgga ggaggggtcc 2760 tcataccccc acttacagct ctctttgagg ggatatgccacactagtcac atggtggacc 2820 ctgagctaga gctgggtctt ggctgggtct tcccctctgtcctattaagc tatggataca 2880 tccacagctt ataccctgta tgagctggag aagaacttacgtatctggag ttactggaag 2940 attgctcttt ttttttttct tctttaaaca ccccctcccccaggtcatca tcttgtttca 3000 gatttttatt caaattctta ttgaaggctg atttttgaataaggagcaga ggagctgttc 3060 tgccacaaat gacccccaaa tgacaggcac tgagactttctttcttcctt ccttccttcc 3120 ttccttcctt ccttccttcc ttccttcctt ccttccttcctttctttctt tctttctttc 3180 tttctttctt tctttctttc tttctttctt tctttcttctttcttcct 3228 6 1035 DNA Homo sapiens 6 cgcatgagga cgcgagtgaa atagaccaaggtggaatttc caagggaaaa gcttcggggt 60 ggttttggtc catttctcca gcgaagaagtagacatggcg agcgactccc cggctcgaag 120 cctggatgaa atagatctct cggctctgagggaccctgca gggatctttg aattggtgga 180 acttgttgga aatggaacat acgggcaagtttataagggt cgtcatgtca aaacgggcca 240 gcttgcagcc atcaaggtta tggatgtcacaggggatgaa gaggaagaaa tcaaacaaga 300 aattaacatg ttgaagaaat attctcatcaccggaatatt gctacatact atggtgcttt 360 tatcaaaaag aacccaccag gcatggatgaccaactttgg ttggtgatgg agttttgtgg 420 tgctggctct gtcaccgacc tgatcaagaacacaaaaggt aacacgttga aagaggagtg 480 gattgcatac atctgcaggg aaatcttacgggggctgagt cacctgcacc agcataaagt 540 gattcatcga gatattaaag ggcaaaatgtcttgctgact gaaaatgcag aagttaaact 600 agtggacttt ggagtcagtg ctcagcttgatcgaacagtg ggcaggagga atactttcat 660 tggaactccc tactggatgg caccagaagttattgcctgt gatgaaaacc cagatgccac 720 atatgatttc aagagtgact tgtggtctttgggtatcacc gccattgaaa tggcagaagg 780 tgctccccct ctctgtgaca tgcaccccatgagagctctc ttcctcatcc cccggaatcc 840 agcgcctcgg ctgaagtcta agaagtggtcaaaaaaaatt ccagtcattt attgagagct 900 gcttggtaaa gaatcacagc cagcgaccagcaacagaaca attgatgaag catccattta 960 tacgagacca acctaatgag cgacaggtccgcattcaact caaggaccat attgatagaa 1020 caaagaagaa gcgag 1035 7 4083 DNAHomo sapiens 7 atggcgagcg actccccggc tcgaagcctg gatgaaatag atctctcggctctgagggac 60 cctgcaggga tctttgaatt ggtggaactt gttggaaatg gaacatacgggcaagtttat 120 aagggtcgtc atgtcaaaac gggccagctt gcagccatca aggttatggatgtcacaggg 180 gatgaagagg aagaaatcaa acaagaaatt aacatgttga agaaatattctcatcaccgg 240 aatattgcta catactatgg tgcttttatc aaaaagaacc caccaggcatggatgaccaa 300 ctttggttgg tgatggagtt ttgtggtgct ggctctgtca ccgacctgatcaagaacaca 360 aaaggtaaca cgttgaaaga ggagtggatt gcatacatct gcagggaaatcttacggggg 420 ctgagtcacc tgcaccagca taaagtgatt catcgagata ttaaagggcaaaatgtcttg 480 ctgactgaaa atgcagaagt taaactagtg gactttggag tcagtgctcagcttgatcga 540 acagtgggca ggaggaatac tttcattgga actccctact ggatggcaccagaagttatt 600 gcctgtgatg aaaacccaga tgccacatat gatttcaaga gtgacttgtggtctttgggt 660 atcaccgcca ttgaaatggc agaaggtgct ccccctctct gtgacatgcaccccatgaga 720 gctctcttcc tcatcccccg gaatccagcg cctcggctga agtctaagaagtggtcaaaa 780 aaattccagt catttattga gagctgcttg gtaaagaatc acagccagcgaccagcaaca 840 gaacaattga tgaagcatcc atttatacga gaccaaccta atgagcgacaggtccgcatt 900 caactcaagg accatattga tagaacaaag aagaagcgag gagaaaaagatgagacagag 960 tatgagtaca gtggaagtga ggaagaagag gaggagaatg actcaggagagcccagctcc 1020 atcctgaatc tgccagggga gtcgacgctg cggagggact ttctgaggctgcagctggcc 1080 aacaaggagc gttctgaggc cctacggagg cagcagctgg agcagcagcagcgggagaat 1140 gaggagcaca agcggcagct gctggccgag cgtcagaagc gcatcgaggagcagaaagag 1200 cagaggcggc ggctggagga gcaacaaagg cgagagaagg agctgcggaagcagcaggag 1260 agggagcagc gccggcacta tgaggagcag atgcgccggg aggaggagaggaggcgtgcg 1320 gagcatgaac aggaatacat caggcgacag ttagaggagg agcagagacagttagagatc 1380 ttgcagcagc agctactgca tgaacaagct ctacttctgg aatataagcgcaaacaattg 1440 gaagaacaga gacaagcaga aagactgcag aggcagctaa agcaagaaagagactactta 1500 gtttcccttc agcatcagcg gcaggagcag aggcctgtgg agaagaagccactgtaccat 1560 tacaaagaag gaatgagtcc tagtgagaag ccagcatggg ccaaggaggtagaagaacgg 1620 tcaaggctca accggcaaag ttcccctgcc atgcctcaca aggttgccaacaggatatct 1680 gaccccaacc tgcccccaag gtcggagtcc ttcagcatta gtggagttcagcctgctcga 1740 acacccccca tgctcagacc agtcgatccc cagatcccac atctggtagctgtaaaatcc 1800 cagggacctg ccttgaccgc ctcccagtca gtgcacgagc agcccacaaagggcctctct 1860 gggtttcagg aggctctgaa cgtgacctcc caccgcgtgg agatgccacgccagaactca 1920 gatcccacct cggaaaatcc tcctctcccc actcgcattg aaaagtttgaccgaagctct 1980 tggttacgac aggaagaaga cattccacca aaggtgcctc aaagaacaacttctatatcc 2040 ccagcattag ccagaaagaa ttctcctggg aatggtagtg ctctgggacccagactagga 2100 tctcaaccca tcagagcaag caaccctgat ctccggagaa ctgagcccatcttggagagc 2160 cccttgcaga ggaccagcag tggcagttcc tccagctcca gcacccctagctcccagccc 2220 agctcccaag gaggctccca gcctggatca caagcaggat ccagtgaacgcaccagagtt 2280 cgagccaaca gtaagtcaga aggatcacct gtgcttcccc atgagcctgccaaggtgaaa 2340 ccagaagaat ccagggacat tacccggccc agtcgaccag ctagctacaaaaaagctata 2400 gatgaggatc tgacggcatt agccaaagaa ctaagagaac tccggattgaagaaacaaac 2460 cgcccaatga agaaggtgac tgattactcc tcctccagtg aggagtcagaaagtagcgag 2520 gaagaggagg aagatggaga gagcgagacc catgatggga cagtggctgtcagcgacata 2580 cccagactga taccaacagg agctccaggc agcaacgagc agtacaatgtgggaatggtg 2640 gggacgcatg ggctggagac ctctcatgcg gacagtttca gcggcagtatttcaagagaa 2700 ggaaccttga tgattagaga gacgtctgga gagaagaagc gatctggccacagtgacagc 2760 aatggctttg ctggccacat caacctccct gacctggtgc agcagagccattctccagct 2820 ggaaccccga ctgagggact ggggcgcgtc tcaacccatt cccaggagatggactctggg 2880 actgaatatg gcatggggag cagcaccaaa gcctccttca ccccctttgtggaccccaga 2940 gtataccaga cgtctcccac tgatgaagat gaagaggatg aggaatcatcagccgcagct 3000 ctgtttacta gcgaacttct taggcaagaa caggccaaac tcaatgaagcaagaaagatt 3060 tcggtggtaa atgtaaaccc aaccaacatt cggcctcata gcgacacaccagaaatcaga 3120 aaatacaaga aacgattcaa ctcagaaata ctttgtgcag ctctgtggggtgtaaacctt 3180 ctggtgggga ctgaaaatgg cctgatgctt ttggaccgaa gtgggcaaggcaaagtctat 3240 aatctgatca accggaggcg atttcagcag atggatgtgc tagagggactgaatgtcctt 3300 gtgacaattt caggaaagaa gaataagcta cgagtttact atctttcatggttaagaaac 3360 agaatactac ataatgaccc agaagtagaa aagaaacaag gctggatcactgttggggac 3420 ttggaaggct gtatacatta taaagttgtt aaatatgaaa ggatcaaatttttggtgatt 3480 gccttaaaga atgctgtgga aatatatgct tgggctccta aaccgtatcataaattcatg 3540 gcatttaagt cttttgcaga tctccagcac aagcctctgc tagttgatctcacggtagaa 3600 gaaggtcaaa gattaaaggt tatttttggt tcacacactg gtttccatgtaattgatgtt 3660 gattcaggaa actcttatga tatctacata ccatctcata ttcagggcaatatcactcct 3720 catgctattg tcatcttgcc taaaacagat ggaatggaaa tgcttgtttgctatgaggat 3780 gagggggtgt atgtaaacac ctatggccgg ataactaagg atgtggtgctccaatgggga 3840 gaaatgccca cgtctgtggc ctacattcat tccaatcaga taatgggctggggcgagaaa 3900 gctattgaga tccggtcagt ggaaacagga catttggatg gagtatttatgcataagcga 3960 gctcaaaggt taaagtttct atgtgaaaga aatgataagg tattttttgcatccgtgcga 4020 tctggaggaa gtagccaagt gtttttcatg accctcaaca gaaattccatgatgaactgg 4080 taa 4083 8 275 PRT Mus musculus 8 Met Gly Asp Pro AlaPro Ala Arg Ser Leu Asp Asp Ile Asp Leu Ser 1 5 10 15 Ala Leu Arg AspPro Ala Gly Ile Phe Glu Leu Val Glu Val Val Gly 20 25 30 Asn Gly Thr TyrGly Gln Val Tyr Lys Gly Arg His Val Lys Thr Gly 35 40 45 Gln Leu Ala AlaIle Lys Val Met Asp Val Thr Glu Asp Glu Glu Glu 50 55 60 Glu Ile Lys GlnGlu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg 65 70 75 80 Asn Ile AlaThr Tyr Tyr Gly Ala Phe Ile Lys Lys Ser Pro Pro Gly 85 90 95 Asn Asp AspGln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser 100 105 110 Val ThrAsp Leu Val Lys Asn Thr Lys Gly Asn Ala Leu Lys Glu Asp 115 120 125 CysIle Ala Tyr Ile Cys Arg Glu Ile Leu Arg Gly Leu Ala His Leu 130 135 140His Ala His Lys Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu 145 150155 160 Leu Thr Glu Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala165 170 175 Gln Leu Asp Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly ThrPro 180 185 190 Tyr Trp Met Ala Pro Glu Val Ile Ala Cys Asp Glu Asn ProAsp Ala 195 200 205 Thr Tyr Asp Tyr Arg Ser Asp Ile Trp Ser Leu Gly IleThr Ala Ile 210 215 220 Glu Met Ala Glu Gly Ala Pro Pro Leu Cys Asp MetHis Pro Met Arg 225 230 235 240 Ala Leu Phe Leu Ile Pro Arg Asn Pro ProPro Arg Leu Lys Ser Lys 245 250 255 Lys Trp Ser Lys Lys Phe Thr Asp PheIle Asp Thr Cys Leu Ile Lys 260 265 270 Thr Tyr Leu 275 9 302 PRT Musmusculus 9 Met Asp Glu Gln Ser Gln Gly Met Gln Gly Pro Pro Val Thr GlnPhe 1 5 10 15 Gln Pro Gln Lys Ala Leu Arg Pro Asp Met Gly Tyr Asn ThrLeu Ala 20 25 30 Asn Phe Arg Ile Glu Lys Lys Ile Gly Arg Gly Gln Phe SerGlu Val 35 40 45 Tyr Arg Ala Ser Cys Leu Leu Asp Gly Val Pro Val Ala LeuLys Lys 50 55 60 Val Gln Ile Phe Asp Leu Met Asp Ala Lys Ala Arg Ala AspCys Ile 65 70 75 80 Lys Glu Ile Asp Leu Leu Lys Gln Leu Asn His Pro AsnVal Ile Lys 85 90 95 Tyr Tyr Ala Ser Phe Ile Glu Asp Asn Glu Leu Asn IleVal Leu Glu 100 105 110 Leu Ala Asp Ala Gly Asp Leu Ser Arg Met Ile LysHis Phe Lys Lys 115 120 125 Gln Lys Arg Leu Ile Pro Glu Arg Thr Val TrpLys Tyr Phe Val Gln 130 135 140 Leu Cys Ser Ala Leu Asp His Met His SerArg Arg Val Met His Arg 145 150 155 160 Asp Ile Lys Pro Ala Asn Val PheIle Thr Ala Thr Gly Val Val Lys 165 170 175 Leu Gly Asp Leu Gly Leu GlyArg Phe Phe Ser Ser Lys Thr Thr Ala 180 185 190 Ala His Ser Leu Val GlyThr Pro Tyr Tyr Met Ser Pro Glu Arg Ile 195 200 205 His Glu Asn Gly TyrAsn Phe Lys Ser Asp Ile Trp Ser Leu Gly Cys 210 215 220 Leu Leu Tyr GluMet Ala Ala Leu Gln Ser Pro Phe Tyr Gly Asp Lys 225 230 235 240 Met AsnLeu Tyr Ser Leu Cys Lys Lys Ile Glu Gln Cys Asp Tyr Pro 245 250 255 ProLeu Pro Ser Asp His Tyr Ser Glu Glu Leu Arg Gln Leu Val Asn 260 265 270Ile Cys Ile Asn Pro Asp Pro Glu Lys Arg Pro Asp Ile Ala Tyr Val 275 280285 Tyr Asp Val Ala Lys Arg Met His Ala Cys Thr Ala Ser Thr 290 295 30010 355 PRT Mus musculus 10 Met Ala Arg Glu Asn Gly Glu Ser Ser Ser SerTrp Lys Lys Gln Ala 1 5 10 15 Glu Asp Ile Lys Lys Ile Phe Glu Phe LysGlu Thr Leu Gly Thr Gly 20 25 30 Ala Phe Ser Glu Val Val Leu Ala Glu GluLys Ala Thr Gly Lys Leu 35 40 45 Phe Ala Val Lys Cys Ile Pro Lys Lys AlaLeu Lys Gly Lys Glu Ser 50 55 60 Ser Ile Glu Asn Glu Ile Ala Val Leu ArgLys Ile Lys His Glu Asn 65 70 75 80 Ile Val Ala Leu Glu Asp Ile Tyr GluSer Pro Asn His Leu Tyr Leu 85 90 95 Val Met Gln Leu Val Ser Gly Gly GluLeu Phe Asp Arg Ile Val Glu 100 105 110 Lys Gly Phe Tyr Thr Glu Lys AspAla Ser Thr Leu Ile Arg Gln Val 115 120 125 Leu Asp Ala Val Tyr Tyr LeuHis Arg Met Gly Ile Val His Arg Asp 130 135 140 Leu Lys Pro Glu Asn LeuLeu Tyr Tyr Ser Gln Asp Glu Glu Ser Lys 145 150 155 160 Ile Met Ile SerAsp Phe Gly Leu Ser Lys Met Glu Gly Lys Gly Asp 165 170 175 Val Met SerThr Ala Cys Gly Thr Pro Gly Tyr Val Ala Pro Glu Val 180 185 190 Leu AlaGln Lys Pro Tyr Ser Lys Ala Val Asp Cys Trp Ser Ile Gly 195 200 205 ValIle Ala Tyr Ile Leu Leu Cys Gly Tyr Pro Pro Phe Tyr Asp Glu 210 215 220Asn Asp Ser Lys Leu Phe Glu Gln Ile Leu Lys Ala Glu Tyr Glu Phe 225 230235 240 Asp Ser Pro Tyr Trp Asp Asp Ile Ser Asp Ser Ala Lys Asp Phe Ile245 250 255 Arg Asn Leu Met Glu Lys Asp Pro Asn Lys Arg Tyr Thr Cys GluGln 260 265 270 Ala Ala Arg His Pro Trp Ile Ala Gly Asp Thr Ala Leu SerLys Asn 275 280 285 Ile His Glu Ser Val Ser Ala Gln Ile Arg Lys Asn PheAla Lys Ser 290 295 300 Lys Trp Arg Gln Ala Phe Asn Ala Thr Ala Val ValArg His Met Arg 305 310 315 320 Arg Leu Gln Leu Gly Ser Ser Leu Asp SerSer Asn Ala Ser Val Ser 325 330 335 Ser Asn Leu Ser Leu Ala Ser Gln LysAsp Cys Ala Ser Gly Thr Phe 340 345 350 His Ala Leu 355 11 631 PRT Musmusculus 11 Met Glu Ser Val Ala Leu Leu Gln Arg Pro Ser Gln Ala Pro SerAla 1 5 10 15 Ser Ala Leu Ala Ser Glu Ser Ala Arg Pro Leu Ala Asp GlyLeu Ile 20 25 30 Lys Ser Pro Lys Pro Leu Met Lys Lys Gln Ala Val Lys ArgHis His 35 40 45 His Lys His Asn Leu Arg His Arg Tyr Glu Phe Leu Glu ThrLeu Gly 50 55 60 Lys Gly Thr Tyr Gly Lys Val Lys Lys Ala Arg Glu Ser SerGly Arg 65 70 75 80 Leu Val Ala Ile Lys Ser Ile Arg Lys Asp Lys Ile LysAsp Glu Gln 85 90 95 Asp Leu Leu His Ile Arg Arg Glu Ile Glu Ile Met SerSer Leu Asn 100 105 110 His Pro His Ile Ile Ala Ile His Glu Val Phe GluAsn Ser Ser Lys 115 120 125 Ile Val Ile Val Met Glu Tyr Ala Ser Arg GlyAsp Leu Tyr Asp Tyr 130 135 140 Ile Ser Glu Arg Pro Arg Leu Ser Glu ArgAsp Ala Arg His Phe Phe 145 150 155 160 Arg Gln Ile Val Ser Ala Leu HisTyr Cys His Gln Asn Gly Ile Val 165 170 175 His Arg Asp Leu Lys Leu GluAsn Ile Leu Leu Asp Ala Asn Gly Asn 180 185 190 Ile Lys Ile Ala Asp PheGly Leu Ser Asn Leu Tyr His Lys Gly Lys 195 200 205 Phe Leu Gln Thr PheCys Gly Ser Pro Leu Tyr Ala Ser Pro Glu Ile 210 215 220 Val Asn Gly LysPro Tyr Val Gly Pro Glu Val Asp Ser Trp Ser Leu 225 230 235 240 Gly ValLeu Leu Tyr Ile Leu Val His Gly Thr Met Pro Phe Asp Gly 245 250 255 GlnAsp His Lys Thr Leu Val Lys Gln Ile Ser Asn Gly Ala Tyr Arg 260 265 270Glu Pro Pro Lys Pro Ser Asp Ala Cys Gly Leu Ile Arg Trp Leu Leu 275 280285 Met Val Asn Pro Thr Arg Arg Ala Thr Leu Glu Asp Val Ala Ser His 290295 300 Trp Trp Val Asn Trp Gly Tyr Thr Thr Gly Val Gly Glu Gln Glu Ala305 310 315 320 Leu Arg Glu Gly Gly His Pro Ser Gly Asp Phe Gly Arg AlaSer Met 325 330 335 Ala Asp Trp Leu Arg Arg Ser Ser Arg Pro Leu Leu GluAsn Gly Ala 340 345 350 Lys Val Cys Ser Phe Phe Lys Gln His Val Pro GlyGly Gly Ser Thr 355 360 365 Val Pro Gly Leu Glu Arg Gln His Ser Leu LysLys Ser Arg Lys Glu 370 375 380 Asn Asp Met Ala Gln Asn Leu Gln Gly AspPro Ala Glu Asp Thr Ser 385 390 395 400 Ser Arg Pro Gly Lys Ser Ser LeuLys Leu Pro Lys Gly Ile Leu Lys 405 410 415 Lys Lys Ser Ser Thr Ser SerGly Glu Val Gln Glu Asp Pro Gln Glu 420 425 430 Leu Arg Pro Val Pro AspThr Pro Gly Gln Pro Val Pro Ala Val Ser 435 440 445 Leu Leu Pro Arg LysGly Ile Leu Lys Lys Ser Arg Gln Arg Glu Ser 450 455 460 Gly Tyr Tyr SerSer Pro Glu Pro Ser Glu Ser Gly Glu Leu Leu Asp 465 470 475 480 Ala SerAsp Val Phe Val Ser Gly Asp Pro Val Glu Gln Lys Ser Pro 485 490 495 GlnAla Ser Gly Leu Leu Leu His Arg Lys Gly Ile Leu Lys Leu Asn 500 505 510Gly Lys Phe Ser Arg Thr Ala Leu Glu Gly Thr Thr Pro Ser Thr Phe 515 520525 Gly Ser Leu Asp Gln Leu Ala Ser Ser His Pro Ala Ala Arg Pro Ser 530535 540 Arg Pro Ser Gly Ala Val Ser Glu Asp Ser Ile Leu Ser Ser Glu Ser545 550 555 560 Phe Asp Gln Leu Asp Leu Pro Glu Arg Leu Pro Glu Thr ProLeu Arg 565 570 575 Gly Cys Val Ser Val Asp Asn Leu Arg Gly Leu Glu GlnPro Pro Ser 580 585 590 Glu Gly Leu Lys Arg Trp Trp Gln Glu Ser Leu GlyAsp Ser Cys Phe 595 600 605 Ser Leu Thr Asp Cys Gln Glu Val Thr Ala AlaTyr Arg Gln Ala Leu 610 615 620 Gly Ile Cys Ser Lys Leu Ser 625 630 12311 PRT Mus musculus 12 Met Ala Gly Pro Ser Trp Gly Leu Pro Arg Leu AspGly Phe Ile Leu 1 5 10 15 Thr Glu Arg Leu Gly Ser Gly Thr Tyr Ala ThrVal Tyr Lys Ala Tyr 20 25 30 Ala Lys Lys Asp Thr Arg Glu Val Val Ala IleLys Cys Val Ala Lys 35 40 45 Lys Ser Leu Asn Lys Ala Ser Val Glu Asn LeuLeu Thr Glu Ile Glu 50 55 60 Ile Leu Lys Gly Ile Arg His Pro His Ile ValGln Leu Lys Asp Phe 65 70 75 80 Gln Trp Asp Asn Asp Asn Ile Tyr Leu IleMet Glu Phe Cys Ala Gly 85 90 95 Gly Asp Leu Ser Arg Phe Ile His Thr ArgArg Ile Leu Pro Glu Lys 100 105 110 Val Ala Arg Val Phe Met Gln Gln LeuAla Ser Ala Leu Gln Phe Leu 115 120 125 His Glu Arg Asn Ile Ser His LeuAsp Leu Lys Pro Gln Asn Ile Leu 130 135 140 Leu Ser Ser Leu Glu Lys ProHis Leu Lys Leu Ala Asp Phe Gly Phe 145 150 155 160 Ala Gln His Met SerPro Trp Asp Glu Lys His Val Leu Arg Gly Ser 165 170 175 Pro Leu Tyr MetAla Pro Glu Met Val Cys Arg Arg Gln Tyr Asp Ala 180 185 190 Arg Val AspLeu Trp Ser Val Gly Val Ile Leu Tyr Glu Ala Leu Phe 195 200 205 Gly GlnPro Pro Phe Ala Ser Arg Ser Phe Ser Glu Leu Glu Glu Lys 210 215 220 IleArg Ser Asn Arg Val Ile Glu Val Arg Leu Ala Gly Ser Arg His 225 230 235240 Pro Pro Gly Ile Glu Gly Leu Lys Ala Gln Lys Phe Val Gln His Cys 245250 255 Ser Ala Gly Ser Gly Arg Phe Met Ala Val Gly His Val Leu Trp Trp260 265 270 Lys Pro Arg Val Trp Ser Val Pro Glu Asp Pro Tyr Gln Pro ArgGln 275 280 285 Ala Thr Asn Asp Gln Ala Gln Ser Ser His Ser Pro Gly LeuGlu Ala 290 295 300 Asn Thr His Leu Ile Gly Asp 305 310 13 266 PRT Homosapiens 13 Met Ala Ser Asp Ser Pro Ala Arg Ser Leu Asp Glu Ile Asp LeuSer 1 5 10 15 Ala Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu Val Glu LeuVal Gly 20 25 30 Asn Gly Thr Tyr Gly Gln Val Tyr Lys Gly Arg His Val LysThr Gly 35 40 45 Gln Leu Ala Ala Ile Lys Val Met Asp Val Thr Gly Asp GluGlu Glu 50 55 60 Glu Ile Lys Gln Glu Ile Asn Met Leu Lys Lys Tyr Ser HisHis Arg 65 70 75 80 Asn Ile Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys AsnPro Pro Gly 85 90 95 Met Asp Asp Gln Leu Trp Leu Val Met Glu Phe Cys GlyAla Gly Ser 100 105 110 Val Thr Asp Leu Ile Lys Asn Thr Lys Gly Asn ThrLeu Lys Glu Glu 115 120 125 Trp Ile Ala Tyr Ile Cys Arg Glu Ile Leu ArgGly Leu Ser His Leu 130 135 140 His Gln His Lys Val Ile His Arg Asp IleLys Gly Gln Asn Val Leu 145 150 155 160 Leu Thr Glu Asn Ala Glu Val LysLeu Val Asp Phe Gly Val Ser Ala 165 170 175 Gln Leu Asp Arg Thr Val GlyArg Arg Asn Thr Phe Ile Gly Thr Pro 180 185 190 Tyr Trp Met Ala Pro GluVal Ile Ala Cys Asp Glu Asn Pro Asp Ala 195 200 205 Thr Tyr Asp Phe LysSer Asp Leu Trp Ser Leu Gly Ile Thr Ala Ile 210 215 220 Glu Met Ala GluGly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg 225 230 235 240 Ala LeuPhe Leu Ile Pro Arg Asn Pro Ala Pro Arg Leu Lys Ser Lys 245 250 255 LysTrp Ser Lys Lys Ile Pro Val Ile Tyr 260 265 14 1360 PRT Homo sapiens 14Met Ala Ser Asp Ser Pro Ala Arg Ser Leu Asp Glu Ile Asp Leu Ser 1 5 1015 Ala Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu Val Glu Leu Val Gly 20 2530 Asn Gly Thr Tyr Gly Gln Val Tyr Lys Gly Arg His Val Lys Thr Gly 35 4045 Gln Leu Ala Ala Ile Lys Val Met Asp Val Thr Gly Asp Glu Glu Glu 50 5560 Glu Ile Lys Gln Glu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg 65 7075 80 Asn Ile Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Asn Pro Pro Gly 8590 95 Met Asp Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser100 105 110 Val Thr Asp Leu Ile Lys Asn Thr Lys Gly Asn Thr Leu Lys GluGlu 115 120 125 Trp Ile Ala Tyr Ile Cys Arg Glu Ile Leu Arg Gly Leu SerHis Leu 130 135 140 His Gln His Lys Val Ile His Arg Asp Ile Lys Gly GlnAsn Val Leu 145 150 155 160 Leu Thr Glu Asn Ala Glu Val Lys Leu Val AspPhe Gly Val Ser Ala 165 170 175 Gln Leu Asp Arg Thr Val Gly Arg Arg AsnThr Phe Ile Gly Thr Pro 180 185 190 Tyr Trp Met Ala Pro Glu Val Ile AlaCys Asp Glu Asn Pro Asp Ala 195 200 205 Thr Tyr Asp Phe Lys Ser Asp LeuTrp Ser Leu Gly Ile Thr Ala Ile 210 215 220 Glu Met Ala Glu Gly Ala ProPro Leu Cys Asp Met His Pro Met Arg 225 230 235 240 Ala Leu Phe Leu IlePro Arg Asn Pro Ala Pro Arg Leu Lys Ser Lys 245 250 255 Lys Trp Ser LysLys Phe Gln Ser Phe Ile Glu Ser Cys Leu Val Lys 260 265 270 Asn His SerGln Arg Pro Ala Thr Glu Gln Leu Met Lys His Pro Phe 275 280 285 Ile ArgAsp Gln Pro Asn Glu Arg Gln Val Arg Ile Gln Leu Lys Asp 290 295 300 HisIle Asp Arg Thr Lys Lys Lys Arg Gly Glu Lys Asp Glu Thr Glu 305 310 315320 Tyr Glu Tyr Ser Gly Ser Glu Glu Glu Glu Glu Glu Asn Asp Ser Gly 325330 335 Glu Pro Ser Ser Ile Leu Asn Leu Pro Gly Glu Ser Thr Leu Arg Arg340 345 350 Asp Phe Leu Arg Leu Gln Leu Ala Asn Lys Glu Arg Ser Glu AlaLeu 355 360 365 Arg Arg Gln Gln Leu Glu Gln Gln Gln Arg Glu Asn Glu GluHis Lys 370 375 380 Arg Gln Leu Leu Ala Glu Arg Gln Lys Arg Ile Glu GluGln Lys Glu 385 390 395 400 Gln Arg Arg Arg Leu Glu Glu Gln Gln Arg ArgGlu Lys Glu Leu Arg 405 410 415 Lys Gln Gln Glu Arg Glu Gln Arg Arg HisTyr Glu Glu Gln Met Arg 420 425 430 Arg Glu Glu Glu Arg Arg Arg Ala GluHis Glu Gln Glu Tyr Ile Arg 435 440 445 Arg Gln Leu Glu Glu Glu Gln ArgGln Leu Glu Ile Leu Gln Gln Gln 450 455 460 Leu Leu His Glu Gln Ala LeuLeu Leu Glu Tyr Lys Arg Lys Gln Leu 465 470 475 480 Glu Glu Gln Arg GlnAla Glu Arg Leu Gln Arg Gln Leu Lys Gln Glu 485 490 495 Arg Asp Tyr LeuVal Ser Leu Gln His Gln Arg Gln Glu Gln Arg Pro 500 505 510 Val Glu LysLys Pro Leu Tyr His Tyr Lys Glu Gly Met Ser Pro Ser 515 520 525 Glu LysPro Ala Trp Ala Lys Glu Val Glu Glu Arg Ser Arg Leu Asn 530 535 540 ArgGln Ser Ser Pro Ala Met Pro His Lys Val Ala Asn Arg Ile Ser 545 550 555560 Asp Pro Asn Leu Pro Pro Arg Ser Glu Ser Phe Ser Ile Ser Gly Val 565570 575 Gln Pro Ala Arg Thr Pro Pro Met Leu Arg Pro Val Asp Pro Gln Ile580 585 590 Pro His Leu Val Ala Val Lys Ser Gln Gly Pro Ala Leu Thr AlaSer 595 600 605 Gln Ser Val His Glu Gln Pro Thr Lys Gly Leu Ser Gly PheGln Glu 610 615 620 Ala Leu Asn Val Thr Ser His Arg Val Glu Met Pro ArgGln Asn Ser 625 630 635 640 Asp Pro Thr Ser Glu Asn Pro Pro Leu Pro ThrArg Ile Glu Lys Phe 645 650 655 Asp Arg Ser Ser Trp Leu Arg Gln Glu GluAsp Ile Pro Pro Lys Val 660 665 670 Pro Gln Arg Thr Thr Ser Ile Ser ProAla Leu Ala Arg Lys Asn Ser 675 680 685 Pro Gly Asn Gly Ser Ala Leu GlyPro Arg Leu Gly Ser Gln Pro Ile 690 695 700 Arg Ala Ser Asn Pro Asp LeuArg Arg Thr Glu Pro Ile Leu Glu Ser 705 710 715 720 Pro Leu Gln Arg ThrSer Ser Gly Ser Ser Ser Ser Ser Ser Thr Pro 725 730 735 Ser Ser Gln ProSer Ser Gln Gly Gly Ser Gln Pro Gly Ser Gln Ala 740 745 750 Gly Ser SerGlu Arg Thr Arg Val Arg Ala Asn Ser Lys Ser Glu Gly 755 760 765 Ser ProVal Leu Pro His Glu Pro Ala Lys Val Lys Pro Glu Glu Ser 770 775 780 ArgAsp Ile Thr Arg Pro Ser Arg Pro Ala Ser Tyr Lys Lys Ala Ile 785 790 795800 Asp Glu Asp Leu Thr Ala Leu Ala Lys Glu Leu Arg Glu Leu Arg Ile 805810 815 Glu Glu Thr Asn Arg Pro Met Lys Lys Val Thr Asp Tyr Ser Ser Ser820 825 830 Ser Glu Glu Ser Glu Ser Ser Glu Glu Glu Glu Glu Asp Gly GluSer 835 840 845 Glu Thr His Asp Gly Thr Val Ala Val Ser Asp Ile Pro ArgLeu Ile 850 855 860 Pro Thr Gly Ala Pro Gly Ser Asn Glu Gln Tyr Asn ValGly Met Val 865 870 875 880 Gly Thr His Gly Leu Glu Thr Ser His Ala AspSer Phe Ser Gly Ser 885 890 895 Ile Ser Arg Glu Gly Thr Leu Met Ile ArgGlu Thr Ser Gly Glu Lys 900 905 910 Lys Arg Ser Gly His Ser Asp Ser AsnGly Phe Ala Gly His Ile Asn 915 920 925 Leu Pro Asp Leu Val Gln Gln SerHis Ser Pro Ala Gly Thr Pro Thr 930 935 940 Glu Gly Leu Gly Arg Val SerThr His Ser Gln Glu Met Asp Ser Gly 945 950 955 960 Thr Glu Tyr Gly MetGly Ser Ser Thr Lys Ala Ser Phe Thr Pro Phe 965 970 975 Val Asp Pro ArgVal Tyr Gln Thr Ser Pro Thr Asp Glu Asp Glu Glu 980 985 990 Asp Glu GluSer Ser Ala Ala Ala Leu Phe Thr Ser Glu Leu Leu Arg 995 1000 1005 GlnGlu Gln Ala Lys Leu Asn Glu Ala Arg Lys Ile Ser Val Val 1010 1015 1020Asn Val Asn Pro Thr Asn Ile Arg Pro His Ser Asp Thr Pro Glu 1025 10301035 Ile Arg Lys Tyr Lys Lys Arg Phe Asn Ser Glu Ile Leu Cys Ala 10401045 1050 Ala Leu Trp Gly Val Asn Leu Leu Val Gly Thr Glu Asn Gly Leu1055 1060 1065 Met Leu Leu Asp Arg Ser Gly Gln Gly Lys Val Tyr Asn LeuIle 1070 1075 1080 Asn Arg Arg Arg Phe Gln Gln Met Asp Val Leu Glu GlyLeu Asn 1085 1090 1095 Val Leu Val Thr Ile Ser Gly Lys Lys Asn Lys LeuArg Val Tyr 1100 1105 1110 Tyr Leu Ser Trp Leu Arg Asn Arg Ile Leu HisAsn Asp Pro Glu 1115 1120 1125 Val Glu Lys Lys Gln Gly Trp Ile Thr ValGly Asp Leu Glu Gly 1130 1135 1140 Cys Ile His Tyr Lys Val Val Lys TyrGlu Arg Ile Lys Phe Leu 1145 1150 1155 Val Ile Ala Leu Lys Asn Ala ValGlu Ile Tyr Ala Trp Ala Pro 1160 1165 1170 Lys Pro Tyr His Lys Phe MetAla Phe Lys Ser Phe Ala Asp Leu 1175 1180 1185 Gln His Lys Pro Leu LeuVal Asp Leu Thr Val Glu Glu Gly Gln 1190 1195 1200 Arg Leu Lys Val IlePhe Gly Ser His Thr Gly Phe His Val Ile 1205 1210 1215 Asp Val Asp SerGly Asn Ser Tyr Asp Ile Tyr Ile Pro Ser His 1220 1225 1230 Ile Gln GlyAsn Ile Thr Pro His Ala Ile Val Ile Leu Pro Lys 1235 1240 1245 Thr AspGly Met Glu Met Leu Val Cys Tyr Glu Asp Glu Gly Val 1250 1255 1260 TyrVal Asn Thr Tyr Gly Arg Ile Thr Lys Asp Val Val Leu Gln 1265 1270 1275Trp Gly Glu Met Pro Thr Ser Val Ala Tyr Ile His Ser Asn Gln 1280 12851290 Ile Met Gly Trp Gly Glu Lys Ala Ile Glu Ile Arg Ser Val Glu 12951300 1305 Thr Gly His Leu Asp Gly Val Phe Met His Lys Arg Ala Gln Arg1310 1315 1320 Leu Lys Phe Leu Cys Glu Arg Asn Asp Lys Val Phe Phe AlaSer 1325 1330 1335 Val Arg Ser Gly Gly Ser Ser Gln Val Phe Phe Met ThrLeu Asn 1340 1345 1350 Arg Asn Ser Met Met Asn Trp 1355 1360 15 25 DNAArtificial Sequence oligonucleotide primer 15 atggcgagcg actccccggctcgaa 25 16 29 DNA Artificial Sequence oligonucleotide primer 16ccagttcatc atggaatttc tgttgaggg 29 17 7 PRT Artificial Sequence peptide17 Leu Arg Arg Ala Ser Leu Gly 1 5 18 13 PRT Artificial Sequence peptide18 Arg Lys Ile Ser Ala Ser Glu Phe Asp Arg Pro Leu Arg 1 5 10 19 18 PRTArtificial Sequence peptide 19 Lys Lys Arg Phe Ser Phe Lys Lys Ser PheLys Leu Ser Gly Phe Ser 1 5 10 15 Phe Lys 20 9 PRT Artificial Sequencepeptide 20 Gln Lys Arg Pro Ser Gln Arg Ser Lys 1 5 21 13 PRT ArtificialSequence peptide 21 Lys Lys Ala Leu Arg Arg Gln Glu Thr Val Asp Ala Leu1 5 10 22 14 PRT Artificial Sequence peptide 22 Pro Leu Ala Arg Thr LeuSer Val Ala Gly Leu Pro Gly Lys 1 5 10 23 10 PRT Artificial Sequencepeptide 23 Arg Arg Arg Asp Asp Asp Ser Asp Asp Asp 1 5 10 24 8 PRTArtificial Sequence peptide 24 Ser Pro Gly Arg Arg Arg Arg Lys 1 5 25 10PRT Artificial Sequence peptide 25 Pro Lys Thr Pro Lys Lys Ala Lys LysLeu 1 5 10 26 9 PRT Artificial Sequence peptide 26 Ala Pro Arg Thr ProGly Gly Arg Arg 1 5 27 20 PRT Artificial Sequence peptide 27 Glu Ala AlaGlu Ala Glu Pro Ala Glu Pro Ser Ser Pro Ala Ala Glu 1 5 10 15 Ala GluGly Ala 20 28 12 PRT Artificial Sequence peptide 28 Leu Met Glu Cys ArgAsn Ser Pro Val Ala Lys Thr 1 5 10 29 18 PRT Artificial Sequence peptide29 Arg Arg Lys Asp Leu His Asp Asp Glu Glu Asp Glu Ala Met Ser Ile 1 510 15 Thr Ala 30 15 PRT Artificial Sequence peptide 30 Leu Ser Ser LeuArg Ala Ser Thr Ser Lys Ser Gly Gly Gln Lys 1 5 10 15 31 13 PRTArtificial Sequence peptide 31 Lys Lys Arg Pro Gln Arg Ala Thr Ser AsnVal Phe Ser 1 5 10 32 18 PRT Artificial Sequence peptide 32 Lys Lys LysLeu Pro Ala Thr Gly Asp Tyr Met Asn Met Ser Pro Val 1 5 10 15 Gly Asp 3311 PRT Artificial Sequence peptide 33 Lys Lys Lys Lys Glu Glu Ile TyrPhe Phe Phe 1 5 10 34 17 PRT Artificial Sequence peptide 34 Ser Gly GlnLeu Ile Asp Ser Met Ala Asn Ser Phe Val Gly Thr Arg 1 5 10 15 Ser 35 12PRT Artificial Sequence peptide 35 Glu Ala Ile Tyr Ala Ala Pro Phe AlaLys Lys Lys 1 5 10 36 7 PRT Artificial Sequence peptide 36 Tyr Ile TyrGly Ser Phe Lys 1 5

What is claimed is:
 1. A method for identifying compounds that inhibitkinase activity comprising: (a) combining a test compound with anisolated polypeptide having kinase activity and a substrate of saidpolypeptide; and (b) determining whether the test compound inhibits thekinase activity of said polypeptide; wherein said polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:10, the amino acid sequence of SEQ ID NO:10 from which one throughfive amino acids have been removed from the N-terminus, the amino acidsequence of SEQ ID NO:10 from which one through five amino acids havebeen removed from the C-terminus, amino acids 23 through 279 of SEQ IDNO:10, and the amino acid sequence of a kinase domain that is at least95% identical to amino acids 23 through 279 of SEQ ID NO:10.
 2. Themethod of claim 1 where the polypeptide is produced by a processcomprising culturing a recombinant host cell, said recombinant host cellcomprising a nucleic acid encoding said polypeptide, under conditionspromoting expression of said polypeptide.
 3. The method of claim 2wherein the nucleic acid comprises a nucleotide sequence selected fromthe group consisting of: (a) SEQ ID NO:3; (b) nucleotides 243 through1307 of SEQ ID NO:3; and (c) a nucleotide sequence encoding an aminoacid sequence selected from the group consisting of SEQ ID NO:10, theamino acid sequence of SEQ ID NO:10 from which one through five aminoacids have been removed from the N-terminus, the amino acid sequence ofSEQ ID NO:10 from which one through five amino acids have been removedfrom the C-tertminus, amino acids 23 through 279 of SEQ ID NO:10, andthe amino acid sequence of a kinase domain that is at least 95%identical to amino acids 23 through 279 of SEQ ID NO:10.
 4. The methodof claim 1 wherein the substrate is a peptide from 8 to 30 amino acidsin length.
 5. The method of claim 1 wherein the test compound iscombined with the polypeptide and the substrate in the presence ofradiolabeled ATP.
 6. A method for identifying compounds that activatekinase activity comprising: (a) combining a test compound with anisolated polypeptide having kinase activity and a substrate of saidpolypeptide; and (b) determining whether the test compound activates thekinase activity of said polypeptide; wherein said polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:10, the amino acid sequence of SEQ ID NO:10 from which one throughfive amino acids have been removed from the N-terminus, the amino acidsequence of SEQ ID NO:10 from which one through five amino acids havebeen removed from the C-terminus, amino acids 23 through 279 of SEQ IDNO:10, and the amino acid sequence of a kinase domain that is at least95% identical to amino acids 23 through 279 of SEQ ID NO:10.
 7. Themethod of claim 6 where the polypeptide is produced by a processcomprising culturing a recombinant host cell, said recombinant host cellcomprising a nucleic acid encoding said polypeptide, under conditionspromoting expression of said polypeptide.
 8. The method of claim 7wherein the nucleic acid comprises a nucleotide sequence selected fromthe group consisting of: (a) SEQ ID NO:3; (b) nucleotides 243 through1307 of SEQ ID NO:3; and (c) a nucleotide sequence encoding an aminoacid sequence selected from the group consisting of SEQ ID NO:10, theamino acid sequence of SEQ ID NO:10 from which one through five aminoacids have been removed from the N-terminus, the amino acid sequence ofSEQ ID NO:10 from which one through five amino acids have been removedfrom the C-terminus, amino acids 23 through 279 of SEQ ID NO:10, and theamino acid sequence of a kinase domain that is at least 95% identical toamino acids 23 through 279 of SEQ ID NO:10.
 9. The method of claim 6wherein the substrate is a peptide from 8 to 30 amino acids in length.10. The method of claim 6 wherein the test compound is combined with thepolypeptide and the substrate in the presence of radiolabeled ATP.
 11. Amethod for identifying compounds that inhibit kinase activitycomprising: (a) combining a test compound with a polypeptide havingkinase activity and a substrate of said polypeptide; and (b) determiningwhether the test compound inhibits the kinase activity of saidpolypeptide; wherein said polypeptide is produced by expression of arecombinant vector comprising a nucleic acid, said nucleic acid encodingan amino acid sequence selected from the group consisting of SEQ IDNO:10, the amino acid sequence of SEQ ID NO:10 from which one throughfive amino acids have been removed from the N-terminus, the amino acidsequence of SEQ ID NO:10 from which one through five amino acids havebeen removed from the C-terminus, amino acids 23 through 279 of SEQ IDNO:10, and the amino acid sequence of a kinase domain that is at least95% identical to amino acids 23 through 279 of SEQ ID NO:10.
 12. Themethod of claim 11 where the test compound is combined with thepolypeptide in a mammalian cell grown in culture.
 13. The method ofclaim 11 where the test compound is combined with the polypeptide invitro.
 14. The method of claim 11 wherein the nucleic acid comprises anucleotide sequence selected from the group consisting of: (a) SEQ IDNO:3; (b) nucleotides 243 through 1307 of SEQ ID NO:3; and (c) anucleotide sequence encoding an amino acid sequence selected from thegroup consisting of SEQ ID NO:10 and amino acids 23 through 279 of SEQID NO:10.
 15. The method of claim 11 wherein the substrate is a peptidefrom 8 to 30 amino acids in length.
 16. The method of claim 11 whereinthe test compound is combined with the polypeptide and the substrate inthe presence of radiolabeled ATP.
 17. The method of claim 16 furthercomprising transferring the radioactive products onto phosphocellulosepaper.
 18. A method for identifying compounds that inhibit kinaseactivity comprising: (a) combining a test compound with an isolatedpolypeptide having kinase activity and a substrate of said polypeptide;and (b) determining whether the test compound inhibits the kinaseactivity of said polypeptide; wherein said polypeptide comprises anamino acid sequence selected from the group consisting of SEQ ID NO:10and amino acids 23 through 279 of SEQ ID NO:10.
 19. The method of claim18 where the polypeptide is produced by a process comprising culturing arecombinant host cell, said recombinant host cell comprising a nucleicacid encoding said polypeptide, under conditions promoting expression ofsaid polypeptide.
 20. The method of claim 19 wherein the nucleic acidcomprises a nucleotide sequence selected from the group consisting of:(a) SEQ ID NO:3; (b) nucleotides 243 through 1307 of SEQ ID NO:3; and(c) a nucleotide sequence encoding an amino acid sequence selected fromthe group consisting of SEQ ID NO:10 and amino acids 23 through 279 ofSEQ ID NO:10.
 21. The method of claim 12 wherein the mammalian cell isselected from the group consisting of dendritic cells, COS-7 cells, CHOcells, and CV1/EBNA cells.
 22. The method of claim 11 where the testcompound is combined with the polypeptide in a cell selected from thegroup consisting of prokaryotic cells, fungal cells, and yeast cells.